COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF Nav1.8 GENE

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the Nav1.8 gene (Nav1.8 gene), comprising an antisense strand having a nucleotide sequence which is less that 25 nucleotides in length and which is substantially complementary to at least a part of the Nav1.8 gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by the expression of the Nav1.8 gene using the pharmaceutical composition; and methods for inhibiting the expression of the Nav1.8 gene gene in a cell.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/593,099, filed Nov. 3, 2006, which claims the benefit ofU.S. Provisional Application No. 60/733,816, filed Nov. 4, 2005; U.S.Provisional Application No. 60/741,586, filed Dec. 2, 2005; U.S.Provisional Application No. 60/763,202, filed Jan. 26, 2006; U.S.Provisional Application No. 60/795,443, filed Apr. 27, 2006; and U.S.Provisional Application No. 60/849,364, filed Oct. 4, 2006. The contentsof these prior applications are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), andits use in mediating RNA interference to inhibit the expression of theNav1.8 gene and the use of the dsRNA to treat pain.

BACKGROUND OF THE INVENTION

Neuropathic pain can be classified as peripheral and central neuropathicpain. Peripheral neuropathic pain is caused by injury or infection ofperipheral sensory nerves, whereas central neuropathic pain is caused bydamage to the CNS and/or the spinal cord. Both peripheral and centralneuropathic pain can occur without obvious initial nerve damage.

A similar definition is given by the International Association for theStudy of Pain (IASP, Seattle, Wash., USA): peripheral neuropathic painis pain initiated or caused by a primary lesion or dysfunction in theperipheral nervous system. Central neuropathic pain is pain initiated orcaused by a primary lesion or dysfunction in the central nervous system.

Peripheral lesions may be lesions of perpherial nerves, e.g. diabeticneuropathy, drug-inducted neuropathy, e.g. upon chemotherapy, lesions ofnerve roots and posterior ganglia, e.g. post-herpetic neuralgia or nerveroot avulsions, neuropathic cancer pain due to compression of peripheralnerves, nerve plexuses and nerve roots, etc. Central lesions may belesions due to infarction, compressive tumors or abscesses, e.g. in thebrainstem, or may be spinal cord lesions due to injury or operations(Jain K K, Emerging Drugs, 2000, 5:241-257; McQuay, 2002, EuropeanJournal of Pain 6 (Suppl. A): 11-18).

The above examples of peripheral and central neuropathic paindemonstrate that peripheral and central neuropathic pain aredistinguished not only by the anatomical location of the lesion ordysfunction, but they also demonstrate that peripheral and centralneuropathic pain can be distinguished by its mechanisms (McQuay, supra).Consequently, there is no clear relation between drug action mechanismand the effect in distinct pain conditions or for single drug classesand different pain conditions (Sindrup S H, Jensen T S, Pain 1999,83:389-400).

Common analgesics like opioids and non-steroidal anti-inflammatory drugs(NSAIDs) improve only insufficiently chronic abnormal pain syndromes asperipheral and central neuropathic pain due to insufficient efficacyand/or dose-limiting side effects. In the search for alternativetreatment regimes to produce satisfactory and sustained pain relief,corticosteroids, conduction blockade, glycerol, anti-convulsants,anti-arrhythmics, antidepressants, local anesthetics, topical agentssuch as capsaicin, gangliosides and electrostimulation have been tried,but only a subset of patients with neuropathic pain respond to suchtreatments and typically, significant pain remains even in theseresponders. The critical issue with current therapies is the therapeuticwindow; a particular treatment might have potential for efficacy but thepatients are not ‘treated to effect’ because of limiting side effectsupon dose escalation.

Central neuropathic pain is a form of neuropathic pain which is aparticularly difficult form to be treated (Yezierski and Burchiel,2002). Due to lesions in the spinothalamocortical pathways, ectopicneuronal discharges can occur in different neurons of the spinal cordand brain. Hyperexcitability in damaged areas of the central nervoussystem plays a major role in the development of central neuropathicpain. Patients with central neuropathic pain almost always havestimulus-independent pain. In addition, in the case of spinal cordinjury, for example, stimulus-dependent pain may be present, usuallybecause skin areas or viscera below the lesions are allodynic. Thus,partial spinal lesions may tend to produce pain to a greater extent thando complete lesions.

Other accepted forms of central neuropathic pain or diseases associatedwith central neuropathic pain exist. Examples include inflammatory CNSdiseases such as multiple sclerosis, myelitis or syphilis, ischemia,hemorrhages or asteriovenous malformations (e.g. post-stroke neuropathicpain) located in the thalamus, spinothalamic pathway or thalamocorticalprojections, and syrnigomyelia (Koltzenburg, Pain 2002—An UpdatedReview: Refresher Course Syllabus; IASP Press, Seattle, 2002).

Na⁺-channels are central to the generation of action potentials in allexcitable cells such as neurons and myocytes. As such they play keyroles in a variety of disease states such as pain (See, Waxman, S. G.,S. Dib-Hajj, et al. (1999) “Sodium channels and pain” Proc Natl Acad SciUSA 96(14): 7635-9 and Waxman, S. G., T. R. Cummins, et al. (2000)“Voltage-gated sodium channels and the molecular pathogenesis of pain: areview” J Rehabil Res Dev 37(5): 517-28). Three members of the genefamily (NaV1.8, 1.9, 1.5) are resistant to block by the well-known Nachannel blocker TTX, demonstrating subtype specificity within this genefamily. Mutational analysis has identified glutamate 387 as a criticalresidue for TTX binding (See, Noda, M., H. Suzuki, et al. (1989) “Asingle point mutation confers tetrodotoxin and saxitoxin insensitivityon the sodium channel II” FEBS Lett 259(1): 213-6).

In general, voltage-gated sodium channels (NaVs) are responsible forinitiating the rapid upstroke of action potentials in excitable tissuein nervous system, which transmit the electrical signals that composeand encode normal and aberrant pain sensations. Antagonists of NaVchannels can attenuate these pain signals and are useful for treating avariety of pain conditions, including but not limited to acute, chronic,inflammatory, and neuropathic pain. Known NaV antagonists include TTX,lidocaine (See Mao, J. and L. L. Chen (2000) “Systemic lidocaine forneuropathic pain relief” Pain 87(1): 7-17.) bupivacaine, carbamazepine,mexilitene, phenyloin (See Jensen, T. S. (2002) “Anticonvulsants inneuropathic pain: rationale and clinical evidence” Eur J Pain 6 (SupplA): 61-8), and lamotrigine (See Rozen, T. D. (2001) “Antiepileptic drugsin the management of cluster headache and trigeminal neuralgia” Headache41 Suppl 1: S25-32 and Jensen, T. S. (2002). However, side effectsinclude dizziness, somnolence, nausea and vomiting (See Tremont-Lukats,I. W., C. Megeff, and M. M. Backonja (2000) “Anticonvulsants forneuropathic pain syndromes: mechanisms of action and place in therapy”Drugs 60:1029-1052) that limit the utility of these known NaVantagonists for the treatment of pain. These side effects are thought toresult at least in part from the block of multiple NaV subtypes. Anagent that inhibits the NaV1.8 channel selectively would provide a muchgreat therapeutic window than these non-selective, known NaVantagonists.

The detection and transmission of nociceptive stimuli is mediated bysmall sensory neurons. Immunohistochemical, in-situ hybridization andelectrophysiology experiments have all shown that the sodium channelNaV1.8 is selectively localized to the small sensory neurons of thedorsal root ganglion and trigeminal ganglion (see Akopian, A. N., L.Sivilotti, et al. (1996) “A tetrodotoxin-resistant voltage-gated sodiumchannel expressed by sensory neurons” Nature 379(6562): 257-62.). Afterexperimental nerve injury, immunohistochemical data demonstrated anaccumulation of Nav1.8 at the site of nerve injury, concomitant with anupregulation in TTX-resistant sodium current, consistent with NaV1.8 asa mechanism underlying hyperalgesia (see Gold, M. S., D. Weinreich, etal. (2003) “Redistribution of Nav1.8 in uninjured axons enablesneuropathic pain” J. Neurosci. 23:158-166). Attenuation of NaV1.8expression with antisense oligodeoxynucleotides administered byintrathecal injection prevented experimental nerve-injury inducedredistribution of Nav1.8 in the sciatic nerve and reversed neuropathicpain (tactile and thermal hyperalgesia), demonstrating a causal role ofNav1.8 in nerve-injury induced pain (see also Lai, J., M. S. Gold, etal. (2002) “Inhibition of neuropathic pain by decreased expression ofthe tetrodotoxin-resistant sodium channel, NaV1.8” Pain 95(1-2): 143-52,and Lai, J., J. C. Hunter, et al. (2000) “Blockade of neuropathic painby antisense targeting of tetrodotoxin-resistant sodium channels insensory neurons” Methods Enzymol 314: 201-13.). In inflammatory painmodels, intrathecal administration of antisense oligodeoxynucleotidesagainst NaV1.8 resulted in a significant reduction in PGE₂-inducedhyperalgesia (see Khasar, S. G., M. S. Gold, et al. (1998) “Atetrodotoxin-resistant sodium current mediates inflammatory pain in therat” Neurosci Lett 256(1): 17-20) and in CFA (complete Freund'sadjuvant)—induced hyperalgesia (Porreca, F., J. Lai, et al. (1999) “Acomparison of the potential role of the tetrodotoxin-insensitive sodiumchannels, PN3/SNS and NaN/SNS2, in rat models of chronic pain” Proc.Nat'l. Acad. Sci. 96: 7640-7644). In addition, in a rat model ofvisceral pain, induced by intravesical infusion of acetic acid, bladderhyperactivity was reduced by intrathecal injection of antisenseoligodeoxynucleotides against NaV1.8, showing that Nav1.8 contributes tothe activation of sensory nerves in visceral pain (Yoshimura, N., S.Seki, et al. (2001) “The involvement of the tetrodotoxin-resistantsodium channel Nav1.8 (PN3/SNS) in a rat model of visceral pain” J.Neurosci. 21: 8690-8696).

Taken together, these data support a role for NaV1.8 in the detectionand transmission of inflammatory and neuropathic pain.

Recently, double-stranded RNA molecules (dsRNA) have been shown to blockgene expression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofthe Nav1.8 gene in C. elegans. dsRNA has also been shown to degradetarget RNA in other organisms, including plants (see, e.g., WO 99/53050,Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see,e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals(see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). Thisnatural mechanism has now become the focus for the development of a newclass of pharmaceutical agents for treating disorders that are caused bythe aberrant or unwanted regulation of a gene.

Despite significant advances in the field of RNAi and advances in thetreatment of pain, there remains a need for an agent that canselectively and efficiently silence the Nav1.8 gene using the cell's ownRNAi machinery that has both high biological activity and in vivostability, and that can effectively inhibit expression of a targetNav1.8 gene for use in treating pain.

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the Nav1.8gene in a cell or mammal using such dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases caused by the expression of the Nav1.8 gene, such as in thepropagation of pain signals in neuropathic and inflammatory pain. ThedsRNA of the invention comprises an RNA strand (the antisense strand)having a region which is less than 30 nucleotides in length and issubstantially complementary to at least part of an mRNA transcript ofthe Nav1.8 gene.

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the Nav1.8 gene.The dsRNA comprises at least two sequences that are complementary toeach other. The dsRNA comprises a sense strand comprising a firstsequence and an antisense strand comprising a second sequence. Theantisense strand comprises a nucleotide sequence which is substantiallycomplementary to at least part of an mRNA encoding Nav1.8, and theregion of complementarity is less than 30 nucleotides in length. ThedsRNA, upon contacting with a cell expressing the Nav1.8, inhibits theexpression of the Nav1.8 gene by at least 20%.

For example, the dsRNA molecules of the invention can be comprised of afirst sequence of the dsRNA that is selected from the group consistingof the sense sequences of Tables 1, 4 and 6 and the second sequence isselected from the group consisting of the antisense sequences of Tables1, 4 and 6. The dsRNA molecules of the invention can be comprised ofnaturally occurring nucleotides or can be comprised of at least onemodified nucleotide, such as a 2′-O-methyl modified nucleotide, anucleotide comprising a 5′-phosphorothioate group, and a terminalnucleotide linked to a cholesteryl derivative or dodecanoic acidbisdecylamide group. Alternatively, the modified nucleotide may bechosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide. Preferably, the first sequence of said dsRNA is selectedfrom the group consisting of the sense sequences of Tables 1, 4 and 6and the second sequence is selected from the group consisting of theantisense sequences of Tables 1, 4 and 6.

In another embodiment, the invention provides a cell comprising one ofthe dsRNAs of the invention. The cell is preferably a mammalian cell,such as a human cell.

In another embodiment, the invention provides a pharmaceuticalcomposition for inhibiting the expression of the Nav1.8 gene in anorganism, comprising one or more of the dsRNA of the invention and apharmaceutically acceptable carrier.

In another embodiment, the invention provides a method for inhibitingthe expression of the Nav1.8 gene in a cell, comprising the followingsteps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA comprises at least two sequences that        are complementary to each, other. The dsRNA comprises a sense        strand comprising a first sequence and an antisense strand        comprising a second sequence. The antisense strand comprises a        region of complementarity which is substantially complementary        to at least a part of an mRNA encoding Nav1.8, and wherein the        region of complementarity is less than 30 nucleotides in length        and wherein the dsRNA, upon contact with a cell expressing the        Nav1.8, inhibits expression of the Nav1.8 gene by at least 20%;        and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of the        Nav1.8 gene, thereby inhibiting expression of the Nav1.8 gene in        the cell.

In another embodiment, the invention provides methods for treating,preventing or managing pain comprising administering to a patient inneed of such treatment, prevention or management a therapeutically orprophylactically effective amount of one or more of the dsRNAs of theinvention.

In another embodiment, the invention provides vectors for inhibiting theexpression of the Nav1.8 gene in a cell, comprising a regulatorysequence operably linked to a nucleotide sequence that encodes at leastone strand of one of the dsRNA of the invention.

In another embodiment, the invention provides a cell comprising a vectorfor inhibiting the expression of the Nav1.8 gene in a cell. The vectorcomprises a regulatory sequence operably linked to a nucleotide sequencethat encodes at least one strand of one of the dsRNA of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. In vitro activity of the dsRNAs provided in Table 1 against mRNAexpression of transfected human Nav1.8 in Cos-7 cells.

FIG. 2. Lack of cross reactivity of Nav1.8 dsRNAs to endogenous Nav1.5mRNA in SW620 cells in vitro.

FIG. 3. In vitro activity of the dsRNAs provided in Table 1 againstendogenous Nav1.8 mRNA in primary cultures of rat dorsal root ganglioncells.

FIG. 4. Dose response of dsRNA AL-DP-6209 against endogenous Nav1.8 mRNAin primary cultures of dorsal root ganglion cells FIG. 5. In vivoefficacy of dsRNA AL-DP-6209 with iFECT against complete Freund'sadjuvant—induced tactile hyperalgesia in rats.

FIG. 6. In vivo efficacy of dsRNA AL-DP-4459 and AL-DP-4461 againstcomplete Freund's adjuvant—induced tactile hyperalgesia in rats.

FIG. 7. In vivo efficacy of dsRNA AL-DP-6050 against complete Freund'sadjuvant—induced tactile hyperalgesia in rats.

FIG. 8. In vivo efficacy of dsRNAs AL-DP-6050 and AL-DP-4459 againstcomplete Freund's adjuvant—induced thermal hyperalgesia in rats.

FIG. 9. In vivo efficacy of dsRNA AL-DP-4459 against spinal nerveligatio—induced tactile and thermal hyperalgesia in rats.

FIG. 10. Stability of unconjugated dsRNAs AL-DP-6050, AL-DP-6209,AL-DP-6217, AL-DP-6218 and AL-DP-6219 in human cerebrospinal fluid at37° C.

FIG. 11. Stability of cholesterol-conjugated dsRNA AL-DP-4459 in humancerebrospinal fluid at 37° C.

FIG. 12. Dose response of siRNA AL-DP-6050 and its cholesterol conjugateAL-DP-4459 against mRNA expression of endogenous Nav1.8 in primarycultures of rat dorsal root ganglion cells.

FIG. 13. In vivo efficacy of dsRNA AL-DP-6050 against spinal nerveligation—induced thermal hyperalgesia in rats.

FIG. 14. In vivo efficacy of ND98-2.7 liposomal formulation of dsRNAAL-DP-6050 against spinal nerve ligation—induced thermal hyperalgesia inrats.

FIG. 15. Structure of ND98 lipid

DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the Nav1.8gene in a cell or mammal using the dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases in a mammal caused by the expression of the Nav1.8 gene usingdsRNA. dsRNA directs the sequence-specific degradation of mRNA through aprocess known as RNA interference (RNAi). The process occurs in a widevariety of organisms, including mammals and other vertebrates.

The dsRNA of the invention comprises an RNA strand (the antisensestrand) having a region which is less than 30 nucleotides in length andwhich is substantially complementary to at least part of an mRNAtranscript of the Nav1.8 gene. The use of these dsRNAs enables thetargeted degradation of mRNAs of genes that are implicated in painresponse in mammals. Using cell-based and animal assays, the presentinventors have demonstrated that very low dosages of these dsRNA canspecifically and efficiently mediate RNAi, resulting in significantinhibition of expression of the Nav1.8 gene. Thus, the methods andcompositions of the invention comprising these dsRNAs are useful fortreating pain.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of atarget Nav1.8 gene, as well as compositions and methods for treatingdiseases and disorders caused by the expression of Nav1.8, such asneuropathic and inflammatory pain. The pharmaceutical compositions ofthe invention comprise a dsRNA having an antisense strand comprising aregion of complementarity which is less than 30 nucleotides in lengthand is substantially complementary to at least part of an RNA transcriptof the Nav1.8 gene, together with a pharmaceutically acceptable carrier.

Accordingly, certain aspects of the invention provide pharmaceuticalcompositions comprising the dsRNA of the invention together with apharmaceutically acceptable carrier, methods of using the compositionsto inhibit expression of the Nav1.8 gene, and methods of using thepharmaceutical compositions to treat diseases caused by expression ofthe Nav1.8 gene.

I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, and uracil may be replacedby other moieties without substantially altering the base pairingproperties of an oligonucleotide comprising a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.

By “Nav1.8” as used herein is meant, any Nav1.8 protein, peptide, orpolypeptide associated with the development or maintenance of an ionchannel. The terms “Nav1.8” also refer to nucleic acid sequencesencoding any Nav1.8 protein, peptide, or polypeptide. For the Examples,the Nav1.8 mRNA sequences used were human (NM_(—)006514), mouse(NM_(—)009134), rat (NM_(—)017247) and dog (NM001003203) mRNA sequences.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the Nav1.8 gene, including mRNA that is a product of RNA processingof a primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to, describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butpreferably not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches witi regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding Nav1.8). For example, a polynucleotide iscomplementary to at least a part of a Nav1.8 mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding Nav1.8.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to aribonucleic acid molecule, or complex of ribonucleic acid molecules,having a duplex structure comprising two anti-parallel and substantiallycomplementary, as defined above, nucleic acid strands. The two strandsforming the duplex structure may be different portions of one larger RNAmolecule, or they may be separate RNA molecules. Where the two strandsare part of one larger molecule, and therefore are connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting RNA chain is referred to as a “hairpin loop”. Where thetwo strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker”. The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA. In addition to the duplex structure, a dsRNA may compriseone or more nucleotide overhangs.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence., the mismatches are most tolerated in the terminal regionsand, if present, are preferably in a terminal region or regions, e.g.,within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of”, in as far as theyrefer to the Nav1.8 gene, herein refer to the at least partialsuppression of the expression of the Nav1.8 gene, as manifested by areduction of the amount of mRNA transcribed from the Nav1.8 gene whichmay be isolated from a first cell or group of cells in which the Nav1.8gene is transcribed and which has or have been treated such that theexpression of the Nav1.8 gene is inhibited, as compared to a second cellor group of cells substantially identical to the first cell or group ofcells but which has or have not been so treated (control cells). Thedegree of inhibition is usually expressed in terms of

${\frac{( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} ) - ( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} )}{( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} )} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to Nav1.8 genetranscription, e.g. the amount of protein encoded by the Nav1.8 genewhich is secreted by a cell, or the number of cells displaying a certainphenotype, e.g apoptosis. In principle, Nav1.8 gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given siRNA inhibitsthe expression of the Nav1.8 gene by a certain degree and therefore isencompassed by the instant invention, the assay provided in the Examplesbelow shall serve as such reference.

For example, in certain instances, expression of the Nav1.8 gene issuppressed by at least about 20%, 25%, 35%, or 50% by administration ofthe double-stranded oligonucleotide of the invention. In a preferredembodiment, the Nav1.8 gene is suppressed by at least about 60%, 70%, or80% by administration of the double-stranded oligonucleotide of theinvention. In a more preferred embodiment, the Nav1.8 gene is suppressedby at least about 85%, 90%, or 95% by administration of thedouble-stranded oligonucleotide of the invention. In a most preferredembodiment, the Nav1.8 gene is suppressed by at least about 98%, 99% ormore by administration of the double-stranded oligonucleotide of theinvention.

The terms “treat”, “treatment”, and the like, refer to relief from oralleviation of the perception of pain, including the relief from oralleviation of the intensity and/or duration of a pain (e.g., burningsensation, tingling, electric-shock-like feelings, etc.) experienced bya subject in response to a given stimulus (e.g., pressure, tissueinjury, cold temperature, etc.). Relief from or alleviation of theperception of pain can be any detectable decrease in the intensity orduration of pain. Treatment can occur in a subject (e.g., a human orcompanion animal) suffering from a pain condition or having one or moresymptoms of a pain-related disorder that can be treated according to thepresent invention, or in an animal model of pain, such as the SNL ratmodel of neuropathic pain or CFA rat model of chronic pain describedherein, or another animal model of pain. In the context of the presentinvention insofar as it relates to any of the other conditions recitedherein below (other than pain), the terms “treat”, “treatment”, and thelike mean to relieve or alleviate at least one symptom associated withsuch condition, or to slow or reverse the progression of such condition.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management of painor an overt symptom of pain. The specific amount that is therapeuticallyeffective can be readily determined by ordinary medical practitioner,and may vary depending on factors known in the art, such as, e.g. thetype of pain, the patient's history and age, the stage of pain, and theadministration of other anti-pain agents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. DOUBLE-STRANDED RIBONUCLEIC ACID (dsRNA)

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the Nav1.8 genein a cell or mammal, wherein the dsRNA comprises an antisense strandcomprising a region of complementarity which is complementary to atleast a part of an mRNA formed in the expression of the Nav1.8 gene, andwherein the region of complementarity is less than 30 nucleotides inlength and wherein said dsRNA, upon contact with a cell expressing saidNav1.8 gene, inhibits the expression of said Nav1.8 gene by at least20%. The dsRNA comprises two RNA strands that are sufficientlycomplementary to hybridize to form a duplex structure. One strand of thedsRNA (the antisense strand) comprises a region of complementarity thatis substantially complementary, and preferably fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of the Nav1.8 gene, the other strand (the sense strand)comprises a region which is complementary to the antisense strand, suchthat the two strands hybridize and form a duplex structure when combinedunder suitable conditions. Preferably, the duplex structure is between15 and 30, more preferably between 18 and 25, yet more preferablybetween 19 and 24, and most preferably between 21 and 23 base pairs inlength. Similarly, the region of complementarity to the target sequenceis between 15 and 30, more preferably between 18 and 25, yet morepreferably between 19 and 24, and most preferably between 21 and 23nucleotides in length. The dsRNA of the invention may further compriseone or more single-stranded nucleotide overhang(s). The dsRNA can besynthesized by standard methods known in the art as further discussedbelow, e.g., by use of an automated DNA synthesizer, such as arecommercially available from, for example, Biosearch, Applied Biosystems,Inc. In a preferred embodiment, the Nav1.8 gene is the human Nav1.8gene. In specific embodiments, the antisense strand of the dsRNAcomprises the antisense sequences of Tables 1, 4 and 6 and the secondsequence is selected from the group consisting of the sense sequences ofTables 1, 4 and 6.

In further embodiments, the dsRNA comprises at least one nucleotidesequence selected from the groups of sequences provided in Tables 1, 4and 6. In other embodiments, the dsRNA comprises at least two sequencesselected from this group, wherein one of the at least two sequences iscomplementary to another of the at least two sequences, and one of theat least two sequences is substantially complementary to a sequence ofan mRNA generated in the expression of the Nav1.8 gene. Preferably, thedsRNA comprises two oligonucleotides, wherein one oligonucleotide isdescribed by Tables 1, 4 and 6 and the second oligonucleotide isdescribed by Tables 1, 4 and 6.

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 1, 4 and 6, the dsRNAs ofthe invention can comprise at least one strand of a length of minimally21 nt. It can be reasonably expected that shorter dsRNAs comprising oneof the sequences of Tables 1, 4 and 6 minus only a few nucleotides onone or both ends may be similarly effective as compared to the dsRNAsdescribed above. Hence, dsRNAs comprising a partial sequence of at least15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of thesequences of Tables 1, 4 and 6, and differing in their ability toinhibit the expression of the Nav1.8 gene in a FACS assay as describedherein below by not more than 5, 10, 15, 20, 25, or 30% inhibition froma dsRNA comprising the full sequence, are contemplated by the invention.

The dsRNA of the invention can contain one or more mismatches to thetarget sequence. In a preferred embodiment, the dsRNA of the inventioncontains no more than 3 mismatches. If the antisense strand of the dsRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of the Nav1.8 gene, the dsRNA preferably doesnot contain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of the Nav1.8 gene. Consideration of the efficacy ofdsRNAs with mismatches in inhibiting expression of the Nav1.8 gene isimportant, especially if the particular region of complementarity in theNav1.8 gene is known to have polymorphic sequence variation within thepopulation.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, preferably 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. Moreover, thepresent inventors have discovered that the presence of only onenucleotide overhang strengthens the interference activity of the dsRNA,without affecting its overall stability. dsRNA having only one overhanghas proven particularly stable and effective in vivo, as well as in avariety of cells, cell culture mediums, blood, and serum. Preferably,the single-stranded overhang is located at the 3′-terminal end of theantisense strand or, alternatively, at the 3′-terminal end of the sensestrand. The dsRNA may also have a blunt end, preferably located at the5′-end of the antisense strand. Such dsRNAs have improved stability andinhibitory activity, thus allowing administration at low dosages, i.e.,less than 5 mg/kg body weight of the recipient per day. Preferably, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids of the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Chemical modifications may include,but are not limited to 2′ modifications, introduction of non-naturalbases, covalent attachment to a ligand, and replacement of phosphatelinkages with thiophosphate linkages. In this embodiment, the integrityof the duplex structure is strengthened by at least one, and preferablytwo, chemical linkages. Chemical linking may be achieved by any of avariety of well-known techniques, for example by introducing covalent,ionic or hydrogen bonds; hydrophobic interactions, van der Waals orstacking interactions; by means of metal-ion coordination, or throughuse of purine analogues. Preferably, the chemical groups that can beused to modify the dsRNA include, without limitation, methylene blue;bifunctional groups, preferably bis-(2-chloroethyl)amine;N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. Inone preferred embodiment, the linker is a hexa-ethylene glycol linker.In this case, the dsRNA are produced by solid phase synthesis and thehexa-ethylene glycol linker is incorporated according to standardmethods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996)35:14665-14670). In a particular embodiment, the 5′-end of the antisensestrand and the 3′-end of the sense strand are chemically linked via ahexaethylene glycol linker. In another embodiment, at least onenucleotide of the dsRNA comprises a phosphorothioate orphosphorodithioate groups. The chemical bond at the ends of the dsRNA ispreferably formed by triple-helix bonds. Table 1 provides examples ofmodified RNAi agents of the invention.

In certain embodiments, a chemical bond may be formed by means of one orseveral bonding groups, wherein such bonding groups are preferablypoly-(oxyphosphinicooxy-1,3-propandiol)- and/or polyethylene glycolchains. In other embodiments, a chemical bond may also be formed bymeans of purine analogs introduced into the double-stranded structureinstead of purines. In further embodiments, a chemical bond may beformed by azabenzene units introduced into the double-strandedstructure. In still further embodiments, a chemical bond may be formedby branched nucleotide analogs instead of nucleotides introduced intothe double-stranded structure. In certain embodiments, a chemical bondmay be induced by ultraviolet light.

In yet another embodiment, the nucleotides at one or both of the twosingle strands may be modified to prevent or inhibit the activation ofcellular enzymes, such as, for example, without limitation, certainnucleases. Techniques for inhibiting the activation of cellular enzymesare known in the art including, but not limited to, 2′-aminomodifications, 2′-fluoro modifications, 2′-alkyl modifications,uncharged backbone modifications, morpholino modifications, 2′-O-methylmodifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995)1:1116-8). Thus, at least one 2′-hydroxyl group of the nucleotides on adsRNA is replaced by a chemical group, preferably by a 2′-fluoro or a2′-O-methyl group. Also, at least one nucleotide may be modified to forma locked nucleotide. Such locked nucleotide contains a methylene orethylene bridge that connects the 2′-oxygen of ribose with the 4′-carbonof ribose. Oligonucleotides containing the locked nucleotide aredescribed in Koshkin, A. A., et al., Tetrahedron (1998), 54: 3607-3630)and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404).Introduction of a locked nucleotide into an oligonucleotide improves theaffinity for complementary sequences and increases the meltingtemperature by several degrees (Braasch, D. A. and D. R. Corey, Chem.Biol. (2001), 8:1-7).

Conjugating a ligand to a dsRNA can enhance its cellular absorption. Incertain instances, a hydrophobic ligand is conjugated to the dsRNA tofacilitate direct permeation of the cellular membrane. Alternatively,the ligand conjugated to the dsRNA is a substrate for receptor-mediatedendocytosis. These approaches have been used to facilitate cellpermeation of antisense oligonucleotides. For example, cholesterol hasbeen conjugated to various antisense oligonucleotides resulting incompounds that are substantially more active compared to theirnon-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid DrugDevelopment 2002, 12, 103. Other lipophilic compounds that have beenconjugated to oligonucleotides include 1-pyrene butyric acid,1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand forreceptor-mediated endocytosis is folic acid. Folic acid enters the cellby folate-receptor-mediated endocytosis. dsRNA compounds bearing folicacid would be efficiently transported into the cell via thefolate-receptor-mediated endocytosis. Li and coworkers report thatattachment of folic acid to the 3′-terminus of an oligonucleotideresulted in an 8-fold increase in cellular uptake of theoligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998,15, 1540. Other ligands that have been conjugated to oligonucleotidesinclude polyethylene glycols, carbohydrate clusters, cross-linkingagents, porphyrin conjugates, and delivery peptides.

In certain instances, conjugation of a cationic ligand tooligonucleotides often results in improved resistance to nucleases.Representative examples of cationic ligands are propylammonium anddimethylpropylammonium. Interestingly, antisense oligonucleotides werereported to retain their high binding affinity to mRNA when the cationicligand was dispersed throughout the oligonucleotide. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103 and referencestherein.

The ligand-conjugated dsRNA of the invention may be synthesized by theuse of a dsRNA that bears a pendant reactive functionality, such as thatderived from the attachment of a linking molecule onto the dsRNA. Thisreactive oligonucleotide may be reacted directly withcommercially-available ligands, ligands that are synthesized bearing anyof a variety of protecting groups, or ligands that have a linking moietyattached thereto. The methods of the invention facilitate the synthesisof ligand-conjugated dsRNA by the use of, in some preferred embodiments,nucleoside monomers that have been appropriately conjugated with ligandsand that may further be attached to a solid-support material. Suchligand-nucleoside conjugates, optionally attached to a solid-supportmaterial, are prepared according to some preferred embodiments of themethods of the invention via reaction of a selected serum-binding ligandwith a linking moiety located on the 5′ position of a nucleoside oroligonucleotide. In certain instances, an dsRNA bearing an aralkylligand attached to the 3′-terminus of the dsRNA is prepared by firstcovalently attaching a monomer building block to a controlled-pore-glasssupport via a long-chain aminoalkyl group. Then, nucleotides are bondedvia standard solid-phase synthesis techniques to the monomerbuilding-block bound to the solid support. The monomer building blockmay be a nucleoside or other organic compound that is compatible withsolid-phase synthesis.

The dsRNA used in the conjugates of the invention may be convenientlyand routinely made through the well-known technique of solid-phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is also known to use similar techniques toprepare other oligonucleotides, such as the phosphorothioates andalkylated derivatives.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. Pat. Nos. 5,138,045and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat.No. 5,212,295, drawn to monomers for the preparation of oligonucleotideshaving chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and5,541,307, drawn to oligonucleotides having modified backbones; U.S.Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and thepreparation thereof through reductive coupling; U.S. Pat. No. 5,457,191,drawn to modified nucleobases based on the 3-deazapurine ring system andmethods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides havingβ-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups may be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2′-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat.Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods ofsynthesizing 2′-fluoro-oligonucleotides.

In the ligand-conjugated dsRNA and ligand-molecule bearingsequence-specific linked nucleosides of the invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide.Oligonucleotide conjugates bearing a variety of molecules such assteroids, vitamins, lipids and reporter molecules, has previously beendescribed (see Manoharan et al., PCT Application WO 93/07883). In apreferred embodiment, the oligonucleotides or linked nucleosides of theinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl,2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of anoligonucleotide confers enhanced hybridization properties to theoligonucleotide. Further, oligonucleotides containing phosphorothioatebackbones have enhanced nuclease stability. Thus, functionalized, linkednucleosides of the invention can be augmented to include either or botha phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-O-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group.

In some preferred embodiments, functionalized nucleoside sequences ofthe invention possessing an amino group at the 5′-terminus are preparedusing a DNA synthesizer, and then reacted with an active esterderivative of a selected ligand. Active ester derivatives are well knownto those skilled in the art. Representative active esters includeN-hydrosuccinimide esters, tetrafluorophenolic esters,pentafluorophenolic esters and pentachlorophenolic esters. The reactionof the amino group and the active ester produces an oligonucleotide inwhich the selected ligand is attached to the 5′-position through alinking group. The amino group at the 5′-terminus can be preparedutilizing a 5′-Amino-Modifier C6 reagent. In a preferred embodiment,ligand molecules may be conjugated to oligonucleotides at the5′-position by the use of a ligand-nucleoside phosphoramidite whereinthe ligand is linked to the 5′-hydroxy group directly or indirectly viaa linker. Such ligand-nucleoside phosphoramidites are typically used atthe end of an automated synthesis procedure to provide aligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.

In one preferred embodiment of the methods of the invention, thepreparation of ligand conjugated oligonucleotides commences with theselection of appropriate precursor molecules upon which to construct theligand molecule. Typically, the precursor is an appropriately-protectedderivative of the commonly-used nucleosides. For example, the syntheticprecursors for the synthesis of the ligand-conjugated oligonucleotidesof the invention include, but are not limited to,2′-aminoalkoxy-5′-ODMT-nucleosides,2′-6-aminoalkylamino-5′-ODMT-nucleosides,5′-6-aminoalkoxy-2′-deoxy-nucleosides,5′-6-aminoalkoxy-2-protected-nucleosides,3′-6-aminoalkoxy-5′-ODMT-nucleosides, and3′-aminoalkylamino-5′-ODMT-nucleosides that may be protected in thenucleobase portion of the molecule. Methods for the synthesis of suchamino-linked protected nucleoside precursors are known to those ofordinary skill in the art.

In many cases, protecting groups are used during the preparation of thecompounds of the invention. As used herein, the term “protected” meansthat the indicated moiety has a protecting group appended thereon. Insome preferred embodiments of the invention, compounds contain one ormore protecting groups. A wide variety of protecting groups can beemployed in the methods of the invention. In general, protecting groupsrender chemical functionalities inert to specific reaction conditions,and can be appended to and removed from such functionalities in amolecule without substantially damaging the remainder of the molecule.

Representative hydroxyl protecting groups, for example, are disclosed byBeaucage et al. (Tetrahedron, 1992, 48:2223-2311). Further hydroxylprotecting groups, as well as other representative protecting groups,are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, andOligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed.,IRL Press, N.Y, 1991.

Examples of hydroxyl protecting groups include, but are not limited to,t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl,diphenylmethyl, p,p′-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate,chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate,p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.

Amino-protecting groups stable to acid treatment are selectively removedwith base treatment, and are used to make reactive amino groupsselectively available for substitution. Examples of such groups are theFmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J.Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p. 1) andvarious substituted sulfonylethyl carbamates exemplified by the Nscgroup (Samukov et al., Tetrahedron Lett., 1994, 35:7821; Verhart andTesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).

Additional amino-protecting groups include, but are not limited to,carbamate protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamideprotecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclicimide protecting groups, such as phthalimido and dithiasuccinoyl.Equivalents of these amino-protecting groups are also encompassed by thecompounds and methods of the invention.

Many solid supports are commercially available and one of ordinary skillin the art can readily select a solid support to be used in thesolid-phase synthesis steps. In certain embodiments, a universal supportis used. A universal support allows for preparation of oligonucleotideshaving unusual or modified nucleotides located at the 3′-terminus of theoligonucleotide. Universal Support 500 and Universal Support II areuniversal supports that are commercially available from Glen Research,22825 Davis Drive, Sterling, Va. For further details about universalsupports see Scott et al., Innovations and Perspectives in solid-phaseSynthesis, 3rd International Symposium, 1994, Ed. Roger Epton, MayflowerWorldwide, 115-124]; Azhayev, A. V. Tetrahedron 1999, 55, 787-800; andAzhayev and Antopolsky Tetrahedron 2001, 57, 4977-4986. In addition, ithas been reported that the oligonucleotide can be cleaved from theuniversal support under milder reaction conditions when oligonucleotideis bonded to the solid support via a syn-1,2-acetoxyphosphate groupwhich more readily undergoes basic hydrolysis. See Guzaev, A. I.;Manoharan, M. J. Am. Chem. Soc. 2003, 125, 2380.

The nucleosides are linked by phosphorus-containing ornon-phosphorus-containing covalent internucleoside linkages. For thepurposes of identification, such conjugated nucleosides can becharacterized as ligand-bearing nucleosides or ligand-nucleosideconjugates. The linked nucleosides having an aralkyl ligand conjugatedto a nucleoside within their sequence will demonstrate enhanced dsRNAactivity when compared to like dsRNA compounds that are not conjugated.

The aralkyl-ligand-conjugated oligonucleotides of the invention alsoinclude conjugates of oligonucleotides and linked nucleosides whereinthe ligand is attached directly to the nucleoside or nucleotide withoutthe intermediacy of a linker group. The ligand may preferably beattached, via linking groups, at a carboxyl, amino or oxo group of theligand. Typical linking groups may be ester, amide or carbamate groups.

Specific examples of preferred modified oligonucleotides envisioned foruse in the ligand-conjugated oligonucleotides of the invention includeoligonucleotides containing modified backbones or non-naturalinternucleoside linkages. As defined here, oligonucleotides havingmodified backbones or internucleoside linkages include those that retaina phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of the invention,modified oligonucleotides that do not have a phosphorus atom in theirintersugar backbone can also be considered to be oligonucleosides.

Specific oligonucleotide chemical modifications are described below. Itis not necessary for all positions in a given compound to be uniformlymodified. Conversely, more than one modifications may be incorporated ina single dsRNA compound or even in a single nucleotide thereof.

Preferred modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acidforms are also included.

Representative United States patents relating to the preparation of theabove phosphorus-atom-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is hereinincorporated by reference.

Preferred modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleosides) havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents relating to the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleoside units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigonucleotide, an oligonucleotide mimetic, that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide-containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained, and are bounddirectly or indirectly to atoms of the amide portion of the backbone.Representative United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found in Nielsen etal., Science, 1991, 254, 1497.

Some preferred embodiments of the invention employ oligonucleotides withphosphorothioate linkages and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂—, and —O—N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

The oligonucleotides employed in the ligand-conjugated oligonucleotidesof the invention may additionally or alternatively comprise nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C), and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases, such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligonucleotides of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Id., pages 276-278) and are presentlypreferred base substitutions, even more particularly when combined with2′-methoxyethyl sugar modifications.

Representative United States patents relating to the preparation ofcertain of the above-noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; and 5,808,027; all of which are herebyincorporated by reference.

In certain embodiments, the oligonucleotides employed in theligand-conjugated oligonucleotides of the invention may additionally oralternatively comprise one or more substituted sugar moieties. Preferredoligonucleotides comprise one of the following at the 2′ position: OH;F; O-, S-, or N-alkyl, O-, S-, or N-alkenyl, or O, S- or N-alkynyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl.Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃,O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. apreferred modification includes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim.Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in U.S. Pat. No. 6,127,533,filed on Jan. 30, 1998, the contents of which are incorporated byreference.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides.

As used herein, the term “sugar substituent group” or “2′-substituentgroup” includes groups attached to the 2′-position of the ribofuranosylmoiety with or without an oxygen atom. Sugar substituent groups include,but are not limited to, fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole andpolyethers of the formula (O-alkyl)_(m), wherein m is 1 to about 10.Preferred among these polyethers are linear and cyclic polyethyleneglycols (PEGs), and (PEG)-containing groups, such as crown ethers andthose which are disclosed by Ouchi et al. (Drug Design and Discovery1992, 9:93); Ravasio et al. (J. Org. Chem. 1991, 56:4329); and Delgardoet. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992,9:249), each of which is hereby incorporated by reference in itsentirety. Further sugar modifications are disclosed by Cook (Anti-painDrug Design, 1991, 6:585-607). Fluoro, O-alkyl, O-alkylamino, O-alkylimidazole, O-alkylaminoalkyl, and alkyl amino substitution is describedin U.S. Pat. No. 6,166,197, entitled “Oligomeric Compounds havingPyrimidine Nucleotide(s) with 2′ and 5′ Substitutions,” herebyincorporated by reference in its entirety.

Additional sugar substituent groups amenable to the invention include2′-SR and 2′-NR₂ groups, wherein each R is, independently, hydrogen, aprotecting group or substituted or unsubstituted alkyl, alkenyl, oralkynyl. 2′-SR Nucleosides are disclosed in U.S. Pat. No. 5,670,633,hereby incorporated by reference in its entirety. The incorporation of2′-SR monomer synthons is disclosed by Hamm et al. (J. Org. Chem., 1997,62:3415-3420). 2′-NR nucleosides are disclosed by Goettingen, M., J.Org. Chem., 1996, 61, 6273-6281; and Polushin et al., Tetrahedron Lett.,1996, 37, 3227-3230. Further representative 2′-substituent groupsamenable to the invention include those having one of formula I or II:

wherein,

E is C₁-C₁₀ alkyl, N(Q₃)(Q₄) or N═C (Q₃)(Q₄); each Q₃ and Q₄ is,independently, H, C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogen protectinggroup, a tethered or untethered conjugate group, a linker to a solidsupport; or Q₃ and Q₄, together, form a nitrogen protecting group or aring structure optionally including at least one additional heteroatomselected from N and O;

q₁ is an integer from 1 to 10;

q₂ is an integer from 1 to 10;

q₃ is 0 or 1;

q₄ is 0, 1 or 2;

each Z₁, Z₂ and Z₃ is, independently, C₄-C₇ cycloalkyl, C₅-C₁₄ aryl orC₃-C₁₅ heterocyclyl, wherein the heteroatom in said heterocyclyl groupis selected from oxygen, nitrogen and sulfur;

Z₄ is OM₁, SM₁, or N(M₁)₂; each M₁ is, independently, H, C₁-C₈ alkyl,C₁-C₈ haloalkyl, C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂; M₂ is H orC₁-C₈ alkyl; and

Z₅ is C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,C₆-C₁₄ aryl, N(Q₃)(Q₄), OQ₃, halo, SQ₃ or CN.

Representative 2′-O-sugar substituent groups of formula I are disclosedin U.S. Pat. No. 6,172,209, entitled “Capped 2′-OxyethoxyOligonucleotides,” hereby incorporated by reference in its entirety.Representative cyclic 2′-O-sugar substituent groups of formula II aredisclosed in U.S. Pat. No. 6,271,358, entitled “RNA Targeted 2′-ModifiedOligonucleotides that are Conformationally Preorganized,” herebyincorporated by reference in its entirety.

Sugars having O-substitutions on the ribosyl ring are also amenable tothe invention. Representative substitutions for ring O include, but arenot limited to, S, CH₂, CHF, and CF₂. See, e.g., Secrist et al.,Abstract 21, Program & Abstracts, Tenth International Roundtable,Nucleosides, Nucleotides and their Biological Applications, Park City,Utah, Sep. 16-20, 1992.

Oligonucleotides may also have sugar mimetics, such as cyclobutylmoieties, in place of the pentofuranosyl sugar. Representative UnitedStates patents relating to the preparation of such modified sugarsinclude, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920; and5,859,221, all of which are hereby incorporated by reference.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide. For example, one additional modification of theligand-conjugated oligonucleotides of the invention involves chemicallylinking to the oligonucleotide one or more additional non-ligandmoieties or conjugates which enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties, such as a cholesterol moiety (Letsingeret al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid(Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), athioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993,3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBSLett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Representative United States patents relating to the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941, each of whichis herein incorporated by reference.

The invention also includes compositions employing oligonucleotides thatare substantially chirally pure with regard to particular positionswithin the oligonucleotides. Examples of substantially chirally pureoligonucleotides include, but are not limited to, those havingphosphorothioate linkages that are at least 75% Sp or Rp (Cook et al.,U.S. Pat. No. 5,587,361) and those having substantially chirally pure(Sp or Rp) alkylphosphonate, phosphoramidate or phosphotriester linkages(Cook, U.S. Pat. Nos. 5,212,295 and 5,521,302).

In certain instances, the oligonucleotide may be modified by anon-ligand group. A number of non-ligand molecules have been conjugatedto oligonucleotides in order to enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide, and proceduresfor performing such conjugations are available in the scientificliterature. Such non-ligand moieties have included lipid moieties, suchas cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994,4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann.N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem.Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov etal., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993,75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such oligonucleotide conjugates have beenlisted above. Typical conjugation protocols involve the synthesis ofoligonucleotides bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction may be performed either with the oligonucleotidestill bound to the solid support or following cleavage of theoligonucleotide in solution phase. Purification of the oligonucleotideconjugate by HPLC typically affords the pure conjugate.

Alternatively, the molecule being conjugated may be converted into abuilding block, such as a phosphoramidite, via an alcohol group presentin the molecule or by attachment of a linker bearing an alcohol groupthat may be phosphitylated.

Importantly, each of these approaches may be used for the synthesis ofligand conjugated oligonucleotides. Aminolinked oligonucleotides may becoupled directly with ligand via the use of coupling reagents orfollowing activation of the ligand as an NHS or pentfluorophenolateester. Ligand phosphoramidites may be synthesized via the attachment ofan aminohexanol linker to one of the carboxyl groups followed byphosphitylation of the terminal alcohol functionality. Other linkers,such as cysteamine, may also be utilized for conjugation to achloroacetyl linker present on a synthesized oligonucleotide.

III. PHARMACEUTICAL COMPOSITIONS COMPRISING dsRNA

In one embodiment, the invention provides pharmaceutical compositionscomprising a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition comprising the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of the Nav1.8 gene, such as neuropathic orinflammatory pain.

In another embodiment, the invention provides pharmaceuticalcompositions comprising at least two dsRNAs, designed to targetdifferent regions of the Nav1.8 gene, and a pharmaceutically acceptablecarrier. In this embodiment, the individual dsRNAs are prepared asdescribed in the preceding section, which is incorporated by referenceherein. One dsRNA can have a nucleotide sequence which is substantiallycomplementary to at least one part of the Nav1.8 gene; additional dsRNAsare prepared, each of which has a nucleotide sequence that issubstantially complementary to different part of the Nav1.8 gene. Themultiple dsRNAs may be combined in the same pharmaceutical composition,or formulated separately. If formulated individually, the compositionscontaining the separate dsRNAs may comprise the same or differentcarriers, and may be administered using the same or different routes ofadministration. Moreover, the pharmaceutical compositions comprising theindividual dsRNAs may be administered substantially simultaneously,sequentially, or at preset intervals throughout the day or treatmentperiod.

The pharmaceutical compositions of the invention are administered indosages sufficient to inhibit expression of the Nav1.8 gene. The presentinventors have found that, because of their improved efficiency,compositions comprising the dsRNA of the invention can be administeredat surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogrambody weight of recipient per day is sufficient to inhibit or completelysuppress expression of the Nav1.8 gene, or alleviate chronic pain.

In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0milligrams per kilogram body weight of the recipient per day, preferablyin the range of 0.1 to 200 micrograms per kilogram body weight per day,more preferably in the range of 0.1 to 100 micrograms per kilogram bodyweight per day, even more preferably in the range of 1.0 to 50micrograms per kilogram body weight per day, and most preferably in therange of 1.0 to 25 micrograms per kilogram body weight per day. Thepharmaceutical composition may be administered once daily, or the dsRNAmay be administered as two, three, four, five, six or more sub-doses atappropriate intervals throughout the day or even using continuousinfusion. In that case, the dsRNA contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage. Thedosage unit can also be compounded for delivery over several days, e.g.,using a conventional sustained release formulation which providessustained release of the dsRNA over a several day period. Sustainedrelease formulations are well known in the art. In this embodiment, thedosage unit contains a corresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pain. Such models are usedfor in vivo testing of dsRNA, as well as for determining atherapeutically effective dose.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, epidural, intrathecal, intracerebroventricular,intraparenchymal (within the peripheral or central nervous system),subcutaneous, transdermal, intranasal, airway (aerosol), rectal, vaginaland topical (including buccal and sublingual) administration. Inpreferred embodiments, the pharmaceutical compositions are administeredintrathecally by continuous infusion such as with a pump, orintrathecally by bolus injection. In other preferred embodiments, thepharmaceutical compositions are administered intravenously by continuousinfusion such as with a pump, or intravenously by bolus injection.

For intrathecal, intracerebroventricular, intramuscular,intraparenchymal (within the peripheral or central nervous system),subcutaneous and intravenous use, the pharmaceutical compositions of theinvention will generally be provided in sterile aqueous solutions orsuspensions, buffered to an appropriate pH and isotonicity. Suitableaqueous vehicles include Ringer's solution and isotonic sodium chloride.In a preferred embodiment, the carrier consists exclusively of anaqueous buffer. In this context, “exclusively” means no auxiliary agentsor encapsulating substances are present which might affect or mediateuptake of dsRNA in the cells that express the Nav1.8 gene. Suchsubstances include, for example, micellar structures, such as liposomesor capsids, as described below. Surprisingly, the present inventors havediscovered that compositions containing only naked dsRNA and aphysiologically acceptable solvent are taken up by cells, where thedsRNA effectively inhibits expression of the Nav1.8 gene. Althoughmicroinjection, lipofection, viruses, viroids, capsids, capsoids, orother auxiliary agents are required to introduce dsRNA into cellcultures, surprisingly these methods and agents are not necessary foruptake of dsRNA in vivo. Aqueous suspensions according to the inventionmay include suspending agents such as cellulose derivatives, sodiumalginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agentsuch as lecithin. Suitable preservatives for aqueous suspensions includeethyl and n-propyl p-hydroxybenzoate.

The pharmaceutical compositions useful according to the invention alsoinclude encapsulated formulations to protect the dsRNA against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems:Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811; PCT publication WO91/06309; and European patent publication EP-A-43075, which areincorporated by reference herein.

Using the small interfering RNA vectors previously described, theinvention also provides devices, systems, and methods for delivery ofsmall interfering RNA to target locations in the nervous system and orthe brain. The envisioned route of delivery is through the use ofimplanted, indwelling, intrathecal, intracerebroventricular orintraparenchymal catheters that provide a means for injecting smallvolumes of fluid containing the dsRNA of the invention directly intolocal nerves or local brain tissue, or into bodily fluids surroundingthese tissues. The proximal end of these catheters may be connected toan implanted, intrathecal or intracerebral access port surgicallyaffixed to the patient's body or cranium, or to an implanted drug pumplocated in the patient's torso.

Alternatively, implantable delivery devices, such as an implantable pumpmay be employed. Examples of the delivery devices within the scope ofthe invention include the Model 8506 investigational device (byMedtronic, Inc. of Minneapolis, Minn.), which can be implantedsubcutaneously in the body or on the cranium, and provides an accessport through which therapeutic agents may be delivered to the nerves orbrain. Delivery occurs through a stereotactically implanted polyurethanecatheter. Two models of catheters that can function with the Model 8506access port include the Model 8770 ventricular catheter by Medtronic,Inc., for delivery to the intracerebral ventricles, which is disclosedin U.S. Pat. No. 6,093,180, incorporated herein by reference, and theIPA1 catheter by Medtronic, Inc., for delivery to the brain tissueitself (i.e., intraparenchymal delivery), disclosed in U.S. Ser. Nos.09/540,444 and 09/625,751, which are incorporated herein by reference.The latter catheter has multiple outlets on its distal end to deliverthe therapeutic agent to multiple sites along the catheter path. Inaddition to the aforementioned device, the delivery of the smallinterfering RNA vectors in accordance with the invention can beaccomplished with a wide variety of devices, including but not limitedto U.S. Pat. Nos. 5,735,814, 5,814,014, and 6,042,579, all of which areincorporated herein by reference. Using the teachings of the inventionand those of skill in the art will recognize that these and otherdevices and systems may be suitable for delivery of small interferingRNA vectors for the treatment of pain in accordance with the invention.

In one such embodiment, the method further comprises the steps ofimplanting a pump outside the body or brain, the pump coupled to aproximal end of the catheter, and operating the pump to deliver thepredetermined dosage of the at least one small interfering RNA or smallinterfering RNA vector through the discharge portion of the catheter. Afurther embodiment comprises the further step of periodically refreshinga supply of the at least one small interfering RNA or small interferingRNA vector to the pump outside said body or brain.

Thus, the invention includes the delivery of small interfering RNAvectors using an implantable pump and catheter, like that taught in U.S.Pat. Nos. 5,735,814 and 6,042,579, and further using a sensor as part ofthe infusion system to regulate the amount of small interfering RNAvectors delivered to the nerves or brain, like that taught in U.S. Pat.No. 5,814,014. Other devices and systems can be used in accordance withthe method of the invention, for example, the devices and systemsdisclosed in U.S. Ser. Nos. 09/872,698 (filed Jun. 1, 2001) and09/864,646 (filed May 23, 2001), which are incorporated herein byreference.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions of the invention lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, the dsRNAs of the invention can be administered incombination with other known agents effective in treatment of pain. Inany event, the administering physician can adjust the amount and timingof dsRNA administration on the basis of results observed using standardmeasures of efficacy known in the art or described herein.

Methods for Treating Diseases Caused by Expression of the Nav1.8 Gene

In one embodiment, the invention provides a method for treating asubject having a pathological condition mediated by the expression ofthe Nav1.8 gene, such as neuropathic or inflammatory pain. In thisembodiment, the dsRNA acts as a therapeutic agent for controlling theexpression of the Nav1.8 protein. The method comprises administering apharmaceutical composition of the invention to the patient (e.g.,human), such that expression of the Nav1.8 gene is silenced. Because oftheir high specificity, the dsRNAs of the invention specifically targetmRNAs of the Nav1.8 gene.

Pain

As used herein, the term “pain” is art recognized and includes a bodilysensation elicited by noxious chemical, mechanical, or thermal stimuli,in a subject, e.g., a mammal such as a human. The term “pain” includeschronic pain such as lower back pain; pain due to arthritis, e.g.,osteoarthritis; joint pain, e.g., knee pain or carpal tunnel syndrome;myofascial pain, and neuropathic pain. The term “pain” further includesacute pain, such as pain associated with muscle strains and sprains;tooth pain; headaches; pain associated with surgery; and pain associatedwith various forms of tissue injury, e.g., inflammation, infection, andischemia.

“Neuropathic pain” refers to pain caused by injury or disease of thecentral or peripheral nervous system. In contrast to the immediate(acute) pain caused by tissue injury, neuropathic pain can develop daysor months after a traumatic injury. Neuropathic pain frequently is longlasting or chronic, and is not limited in duration to the period oftissue repair. Neuropathic pain can occur spontaneously, or as a resultof stimulation that normally is not painful. Neuropathic pain is causedby aberrant somatosensory processing, and is associated with chronicsensory disturbances, including spontaneous pain, hyperalgesia (i.e.,sensation of more pain than the stimulus would warrant) and allodynia(i.e., a condition in which ordinarily painless stimuli induce theexperience of pain). Neuropathic pain includes, but is not limited to,pain caused by peripheral nerve trauma, viral infection, diabetesmellitus, chemotherapy, causalgia, plexus-avulsion, spinal cord injury,neuroma, limb amputation, vasculitis, nerve damage from surgery, nervedamage from chronic alcoholism, hypothyroidism, uremia, and vitamindeficiencies, among other causes. Neuropathic pain is one type of painassociated with cancer. Cancer pain can also be “nociceptive” or“mixed.”

“Chronic pain” can be defined as pain lasting longer than three months(Bonica, Semin. Anesth. 1986, 5:82-99), and may be characterized byunrelenting persistent pain that is not fully amenable to routine paincontrol methods. Chronic pain includes, but is not limited to,inflammatory pain, post-operative pain, cancer pain, osteoarthritis painassociated with metastatic cancer, chemotherapy-induced pain, trigeminalneuralgia, acute herpetic and post-herpetic neuralgia, diabeticneuropathy, pain due to arthritis, joint pain, myofascial pain,causalgia, brachial plexus avulsion, occipital neuralgia, reflexsympathetic dystrophy, fibromyalgia, gout, phantom limb pain, burn pain,pain associated with spinal cord injury, multiple sclerosis, reflexsympathetic dystrophy and lower back pain and other forms of neuralgia,neuropathic, and idiopathic pain syndromes.

“Nociceptive pain” is due to activation of pain-sensitive nerve fibers,either somatic or visceral. Nociceptive pain is generally a response todirect tissue damage. The initial trauma causes the release of severalchemicals including bradykinin, serotonin, substance P, histamine, andprostaglandin. When somatic nerves are involved, the pain is typicallyexperienced as an aching or pressure-like sensation.

In the phrase “pain and related disorders”, the term “related disorders”refers to disorders that either cause or are associated with pain, orhave been shown to have similar mechanisms to pain. These disordersinclude addiction, seizure, stroke, ischemia, a neurodegenerativedisorder, anxiety, depression, headache, asthma, rheumatic disease,osteoarthritis, retinopathy, inflammatory eye disorders, pruritis,ulcer, gastric lesions, uncontrollable urination, an inflammatory orunstable bladder disorder, inflammatory bowel disease, irritable bowelsyndrome (IBS), irritable bowel disease (IBD), gastroesophageal refluxdisease (GERD), functional dyspepsia, functional chest pain of presumedoesophageal origin, functional dysphagia, non-cardiac chest pain,symptomatic gastroesophageal disease, gastritis, aerophagia, functionalconstipation, functional diarrhea, burbulence, chronic functionalabdominal pain, recurrent abdominal pain (RAP), functional abdominalbloating, functional biliary pain, functional incontinence, functionalano-rectal pain, chronic pelvic pain, pelvic floor dyssenergia,unspecified functional ano-rectal disorder, cholecystalgia, interstitialcystitis, dysmenorrhea, and dyspareunia.

The invention thus provides the use of an anti-Nav1.8 dsRNA administeredto a human, particularly by intrathecal infusion or injection, or byintravenous infusion or injection, for the treatment of pain, includingneuropathic and inflammatory pain.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, epidural, intrathecal, intracerebroventricular,intraparenchymal (within the peripheral or central nervous system),subcutaneous, transdermal, intranasal, airway (aerosol), nasal, rectal,vaginal and topical (including buccal and sublingual) administration,and epidural administration. In preferred embodiments, thepharmaceutical compositions are administered intrathecally by continuousinfusion such as with a pump, or intrathecally by bolus injection. Inother preferred embodiments, the pharmaceutical compositions areadministered intravenously by continuous infusion such as with a pump,or intravenously by bolus injection.

Methods for Inhibiting Expression of the Nav1.8 Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of the Nav1.8 gene in a mammal. The method comprisesadministering a composition of the invention to the mammal such thatexpression of the target Nav1.8 gene is silenced. Because of their highspecificity, the dsRNAs of the invention specifically target RNAs(primary or processed) of the target Nav1.8 gene. Compositions andmethods for inhibiting the expression of these Nav1.8 genes using dsRNAscan be performed as described elsewhere herein.

In one embodiment, the method comprises administering a compositioncomprising a dsRNA, wherein the dsRNA comprises a nucleotide sequencewhich is complementary to at least a part of an RNA transcript of theNav1.8 gene of the mammal to be treated. When the organism to be treatedis a mammal such as a human, the composition may be administered by anymeans known in the art including, but not limited to oral or parenteralroutes, including intravenous, intramuscular, intraperitoneal, epidural,intrathecal, intracerebroventricular, intraparenchymal (within theperipheral or central nervous system), subcutaneous, transdermal,intranasal, airway (aerosol), rectal, vaginal and topical (includingbuccal and sublingual) administration. In preferred embodiments, thecompositions are administered by intrathecal infusion or injection, orby intravenous infusion or injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES Example 1 Gene Walking of the Nav1.8 Gene

siRNAs were identified in a multi step sequence analysis process inorder to design siRNAs targeting the Nav1.8 gene in 4 species ofinterest.

ClustalW multiple alignment function of BioEdit Sequence AlignmentEditor (version 7.0.4.1) was used to generate a global alignment ofhuman (NM_(—)006514), mouse (NM_(—)009134), rat (NM_(—)017247) and dog(NM001003203) Nav1.8 mRNA sequences.

Conserved regions were identified by embedded sequence analysis functionof the software. Conserved regions were defined as sequence stretcheswith a minimum length of 19 bases for all aligned sequences containingno internal gaps. Sequence positions of conserved regions were countedaccording to the human sequence.

The siRNA design web interface at Whitehead Institute for BiomedicalResearch (http://jura.wi.mit.edu/siRNAext/) was used to identify allpotential siRNAs targeting the conserved regions as well as theirrespective off-target hits to sequences in the human, mouse and ratRefSeq database. siRNAs satisfying the crossreactivity criteria wereselected out of the candidates pool and subjected to the softwareembedded off-target analysis. For this, all selected siRNAs wereanalyzed in 3 rounds by the NCBI blast algorithm against the NCBI human,mouse and rat RefSeq database.

Blast results were downloaded and analyzed by a perl script in order toextract the identity of the best off-target hit for the antisense strainas well as the positions of occurring mismatches.

All siRNA candidates were ranked according to predicted properties. Forthis, different criteria were applied in order to identify siRNAs withthe following properties:

reactivity criterium: targeting human, mouse, rat and dog sequences

specificity criteria: highly specific for human, at least specific forrat and mouse

The 2 siRNAs that satisfied the applied criteria were referred to as“multi-species targeting siRNAs”.

In order to identify more siRNAs a second round of siRNA identificationsteps were conducted correspondingly with additional regions that hadbeen previously eliminated due to missing reactivity with dog sequences.

The resulting pool of 10 siRNAs matching the above mentioned criteriawere referred to as ‘human/rat/mouse targeting siRNAs’.

A third round of the siRNA design process was conducted whiledisregarding cross-reactivity to mouse.

All candidate siRNAs were again extracted and ranked in 3 stepsaccording to the following criteria:

Step 1:

reactivity criterium: targeting human, rat and mouse sequences

specificity criterium: highly specific for human and rat, moderatelyspecific for mouse

4 siRNAs (added to pool of ‘human/rat/mouse targeting siRNAs’)

Step 2:

reactivity criterium: targeting human and rat sequences

sequence-embedded criterium: absense of stretches with more than 3 Gs ina row

specificity criterium: highly specific for human

19 siRNAs

Step 3:

reactivity criterium: targeting human and rat sequences

specificity criterium: highly specific for rat, specific for human andfavorable ddG value

1 siRNA

The pool resulting form steps 2 and 3 (20 siRNAs) were referred to as‘human/rat targeting siRNAs’.

The in silico selected 36 siRNAs were synthesized (Table 1).

Additional sequence selections were performed using the abovegeneralized methods except that cross reactivity between species was notused as a selection criterea: the sequence selection was based solely onthe human Nav1.8 sequence. In addition, ranking was based on off-targetscores based on the closest FASTA hit as was as the number of potentialoff target genes. These siRNAs are provided in Table 4.

TABLE 1 siRNAs specific for Nav 1.8 SEQ SEQ ID antisense strand sequenceID duplex name sense strand sequence (5′-3′) NO: (5′-3′) NO: AL-DP-6042cmcmcmggaumumumumaacmumacmacmcmTT 1 ggugumaguumaaaauccgggTT 2 AL-DP-6043cmcmggaumumumumaacmumacmacmcmaTT 3 uggugumaguumaaaauccggTT 4 AL-DP-6044gacmaacmcmcmggaumumumumaacmumTT 5 aguumaaaauccggguugucTT 6 AL-DP-6045cmcmumumacmaacmcmagcmgcmaggaTT 7 uccugcgcugguugumaaggTT 8 AL-DP-6046aacmcmcmggaumumumumaacmumacmaTT 9 ugumaguumaaaauccggguuTT 10 AL-DP-6047umgumgcmaumgacmcmcmgaacmumgaTT 11 ucmaguucgggucmaugcmacmaTT 12AL-DP-6048 acmaacmcmagcmgcmaggaumgumcmTT 13 gacmauccugcgcugguuguTT 14AL-DP-6049 acmcmcmggaumumumumaacmumacmacmTT 15 gugumaguumaaaauccggguTT16 AL-DP-6050 cmaumcmcmumaumgaacmcmaaumagcmTT 17gcumauugguucmaumaggaugTT 18 AL-DP-6051 cmumumacmaacmcmagcmgcmaggaumTT 19auccugcgcugguugumaagTT 20 AL-DP-6052 umumumaacmumacmacmcmagcmumumumgTT21 cmaaagcuggugumaguumaaaTT 22 AL-DP-6053 gumgumgcmaumgacmcmcmgaacmumgTT23 cmaguucgggucmaugcmacmacTT 24 AL-DP-6202cmggaumumumumaacmumacmacmcmagTT 25 cuggugumaguumaaaauccgTT 26 AL-DP-6203umacmaacmcmagcmgcmaggaumgumTT 27 acmauccugcgcugguugumaTT 28 AL-DP-6204ggumcmumcmumgumgcmcmcmaumumgcmumTT 29 agcmaaugggcmacmagagaccTT 30AL-DP-6205 cmcmcmaagumumcmumaumggumgagcmTT 31 gcucmaccmaumagaacuugggTT32 AL-DP-6206 cmaagumumcmumaumggumgagcmumcmTT 33gagcucmaccmaumagaacuugTT 34 AL-DP-6207 cmumacmagcmacmacmacmcmggacmaTT 35uguccggugugugcugumagTT 36 AL-DP-6208 aagumumcmumaumggumgagcmumcmcmTT 37ggagcucmaccmaumagaacuuTT 38 AL-DP-6209 umumumumgumcmumaaaumgagumumcmaTT39 ugaacucmauuumagacmaaaaTT 40 AL-DP-6210cmcmumcmumcmacmumgumumcmcmgcmcmumcmTT 41 gaggcggaacmagugagaggTT 42AL-DP-6211 aacmcmagumumcmumumumgumggcmcmgTT 43 cggccmacmaaagaacugguuTT44 AL-DP-6212 cmumcmacmumgumumcmcmgcmcmumcmaumgTT 45cmaugaggcggaacmagugagTT 46 AL-DP-6213 cmcmaagumumcmumaumggumgagcmumTT 47agcucmaccmaumagaacuuggTT 48 AL-DP-6214cmagumumcmumumumgumggcmcmgumcmumTT 49 agacggccmacmaaagaacugTT 50AL-DP-6215 ggcmumggcmaggumgcmgcmaagaTT 51 ucuugcgcmaccugccmagccTT 52AL-DP-6216 cmumcmcmumcmumgagggcmagcmacmgTT 53 cgugcugcccucmagaggagTT 54AL-DP-6217 gumcmumumcmacmumgumcmaumumumacmaTT 55 ugumaaaugacmagugaagacTT56 AL-DP-6218 cmaacmcmagcmgcmaggaumgumcmumTT 57 agacmauccugcgcugguugTT58 AL-DP-6219 agaagumaumcmumgaumcmumgggaTT 59 ucccmagaucmagaumacuucuTT60 AL-DP-6220 umcmumacmagcmacmacmacmcmggacmTT 61 guccggugugugcugumagaTT62 AL-DP-6221 agumumcmumaumggumgagcmumcmcmcmTT 63gggagcucmaccmaumagaacuTT 64 AL-DP-6222 umcmumaumggumgagcmumcmcmcmagcmTT65 gcugggagcucmaccmaumagaTT 66 AL-DP-6223agumumcmumumumgumggcmcmgumcmumumTT 67 aagacggccmacmaaagaacuTT 68AL-DP-6224 umcmacmumgumumcmcmgcmcmumcmaumgaTT 69 ucmaugaggcggaacmagugaTT70 AL-DP-6225 ggaumumumumaacmumacmacmcmagcmTT 71 gcuggugumaguumaaaauccTT72

TABLE 4 Further siRNAs specific for Nav1.8 SEQ SEQ Remaining Luc duplexSense strand ID Antisense strand ID activity [% name sequence (5′-3′)NO: sequence (5′-3′) NO: of controls] AD-11159 ccgcuugcugcgcguauucTT 73gaauacgcgcagcaagcggTT 74  26 ± 2 AD-11160 cuugcuaccguaucguggaTT 75uccacgauacgguagcaagTT 76  52 ± 4 AD-11161 gacuucaucgcuaauccgaTT 77ucggauuagcgaugaagucTT 78  29 ± 4 AD-11162 uggauuuuagcgucauuacTT 79guaaugacgcuaaaauccaTT 80  70 ± 6 AD-11163 cgcuugcugcgcguauucaTT 81ugaauacgcgcagcaagcgTT 82  31 ± 5 AD-11164 caacuuccgucgcuuuacuTT 83aguaaagcgacggaaguugTT 84  24 ± 2 AD-11165 gucgcuuuacuccggagucTT 85gacuccggaguaaagcgacTT 86  20 ± 3 AD-11166 aaugaguucacguaccugaTT 87ucagguacgugaacucauuTT 88  17 ± 3 AD-11167 agacuugcuaccguaucguTT 89acgauacgguagcaagucuTT 90  38 ± 2 AD-11168 aguuuauuuauuacggucaTT 91ugaccguaauaaauaaacuTT 92  63 ± 7 AD-11169 aaaugaguucacguaccugTT 93cagguacgugaacucauuuTT 94  46 ± 4 AD-11170 ugucucggcauucgaugcaTT 95ugcaucgaaugccgagacaTT 96  72 ± 5 AD-11171 ucaaagcccuucgaacccuTT 97aggguucgaagggcuuugaTT 98  96 ± 4 AD-11172 gcgggcucuuucucgauuuTT 99aaaucgagaaagagcccgcTT 100  34 ± 4 AD-11173 ccuuguaccuuugucgauuTT 101aaucgacaaagguacaaggTT 102  17 ± 2 AD-11174 gugguucucuccauugcgaTT 103ucgcaauggagagaaccacTT 104  24 ± 3 AD-11175 caaaaggccuaucggagcuTT 105agcuccgauaggccuuuugTT 106  46 ± 3 AD-11176 aaaggccuaucggagcuauTT 107auagcuccgauaggccuuuTT 108  48 ± 5 AD-11177 ggugcaucaacuauaccgaTT 109ucgguauaguugaugcaccTT 110  21 ± 2 AD-11178 aacuaccguaacaaccgaaTT 111uucgguuguuacgguaguuTT 112  42 ± 8 AD-11179 ucgcuuuacuccggagucaTT 113ugacuccggaguaaagcgaTT 114  17 ± 1 AD-11180 gaaaacgccgggcuagucaTT 115ugacuagcccggcguuuucTT 116  36 ± 8 AD-11181 accgaaaaaauaucuccgcTT 117gcggagauauuuuuucgguTT 118 105 ± 15 AD-11182 uucaucgcuaauccgacugTT 119cagucggauuagcgaugaaTT 120  87 ± 14 AD-11183 uccgucgcuuuacuccggaTT 121uccggaguaaagcgacggaTT 122  33 ± 3 AD-11184 gcuuuacuccggagucacuTT 123agugacuccggaguaaagcTT 124  14 ± 1 AD-11185 ggaaaacgccgggcuagucTT 125gacuagcccggcguuuuccTT 126  50 ± 5 AD-11186 acuaccguaacaaccgaaaTT 127uuucgguuguuacgguaguTT 128  41 ± 5 AD-11187 uggccaucguaccaaacagTT 129cuguuugguacgauggccaTT 130  85 ± 7 AD-11188 acuugcuaccguaucguggTT 131ccacgauacgguagcaaguTT 132 109 ± 15 AD-11189 gauaagucucacagcgaagTT 133cuucgcugugagacuuaucTT 134  29 ± 4 AD-11190 auaagucucacagcgaagaTT 135ucuucgcugugagacuuauTT 136  34 ± 4 AD-11191 aagcccuucgaacccuucgTT 137cgaaggguucgaagggcuuTT 138  86 ± 10 AD-11192 agcccuucgaacccuucgcTT 139gcgaaggguucgaagggcuTT 140  98 ± 16 AD-11193 cccuucgaacccuucgcgcTT 141gcgcgaaggguucgaagggTT 142  64 ± 4 AD-11194 aacccaaucgaaauauacuTT 143aguauauuucgauuggguuTT 144  40 ± 4 AD-11195 cgcuuuacuccggagucacTT 145gugacuccggaguaaagcgTT 146  17 ± 2 AD-11196 gaccauuucccgguuuaguTT 147acuaaaccgggaaauggucTT 148  77 ± 4 AD-11197 cgguuuagugccacucgggTT 149cccgaguggcacuaaaccgTT 150  58 ± 6 AD-11198 cacagcaauagaucuccguTT 151acggagaucuauugcugugTT 152  34 ± 4 AD-11199 acuagggauugacacaaccTT 153gguugugucaaucccuaguTT 154  86 ± 3 AD-11200 cccacaauggaucaccuuuTT 155aaaggugauccauugugggTT 156  22 ± 0 AD-11201 ugucuuuucuaggccucgcTT 157gcgaggccuagaaaagacaTT 158  91 ± 10 AD-11202 agcugucgaugucucggcaTT 159ugccgagacaucgacagcuTT 160  42 ± 2 AD-11203 gucucggcauucgaugcagTT 161cugcaucgaaugccgagacTT 162  62 ± 5 AD-11204 ucaaaaucauugccuucgaTT 163ucgaaggcaaugauuuugaTT 164  42 ± 7 AD-11205 cccgcuggcacaugcacgaTT 165ucgugcaugugccagcgggTT 166  42 ± 4 AD-11206 ucauugucuuccguauccuTT 167aggauacggaagacaaugaTT 168  79 ± 11 AD-11207 uuggccaucguaccaaacaTT 169uguuugguacgauggccaaTT 170  59 ± 8 AD-11208 gcacgguggacugccuagaTT 171ucuaggcaguccaccgugcTT 172  95 ± 12 AD-11209 gcccuucgaacccuucgcgTT 173cgcgaaggguucgaagggcTT 174  48 ± 4 AD-11210 ugcgggcucuuucucgauuTT 175aaucgagaaagagcccgcaTT 176  35 ± 5 AD-11211 gaggugcaucaacuauaccTT 177gguauaguugaugcaccucTT 178  36 ± 4 AD-11212 caucaacuauaccgauggaTT 179uccaucgguauaguugaugTT 180  58 ± 4 AD-11213 aaaucauccuaugaaccaaTT 181uugguucauaggaugauuuTT 182  26 ± 2 AD-11214 aaggccuaucggagcuaugTT 183cauagcuccgauaggccuuTT 184  63 ± 4 AD-11215 uguacucccagacaaaucuTT 185agauuugucugggaguacaTT 186  40 ± 3 AD-11216 aggacaucuagcucaauacTT 187guauugagcuagauguccuTT 188  29 ± 2 AD-11217 ccgguuuagugccacucggTT 189ccgaguggcacuaaaccggIT 190  22 ± 2 AD-11218 caucgcuaauccgacugugTT 191cacagucggauuagcgaugTT 192  51 ± 2 AD-11219 aaacuaccguaacaaccgaTT 193ucgguuguuacgguaguuuTT 194  29 ± 3 AD-11220 aucgcuaauccgacuguguTT 195acacagucggauuagcgauTT 196  52 ± 1 AD-11221 cccgguuuagugccacucgTT 197cgaguggcacuaaaccgggIT 198  60 ± 1 AD-11222 uuuagcgucauuacccuggTT 199ccaggguaaugacgcuaaaTT 200 105 ± 3 AD-11223 aauaagcgaggcacuucugTT 201cagaagugccucgcuuauuTT 202  66 ± 5 AD-11224 cuaccguaacaaccgaaaaTT 203uuuucgguuguuacgguagTT 204  25 ± 3 AD-11225 ucgcuaauccgacugugugTT 205cacacagucggauuagcgaTT 206  70 ± 5 AD-11226 ucccucgaaacuaacaacuTT 207aguuguuaguuucgagggaTT 208  24 ± 3 AD-11227 cuuccgucgcuuuacuccgTT 209cggaguaaagcgacggaagTT 210  31 ± 0 AD-11228 uuucccgguuuagugccacTT 211guggcacuaaaccgggaaaTT 212 101 ± 2 AD-11229 gguuuagugccacucgggcTT 213gcccgaguggcacuaaaccTT 214  67 ± 1 AD-11230 acaacccggauuuuaacuaTT 215uaguuaaaauccggguuguTT 216  48 ± 3 AD-11231 cuagggauugacacaaccuTT 217agguugugucaaucccuagTT 218  39 ± 2 AD-11232 ugucgauugugaauaacaaTT 219uuguuauucacaaucgacaTT 220  25 ± 3 AD-11233 caucgugaccagacaagcuTT 221agcuugucuggucacgaugTT 222  47 ± 4 AD-11234 ccuaucggagcuaugugcuTT 223agcacauagcuccgauaggTT 224  58 ± 7 AD-11235 uuagcgucauuacccuggcTT 225gccaggguaaugacgcuaaTT 226  92 ± 7 AD-11236 agcaauagaucuccgugggTT 227cccacggagaucuauugcuTT 228  83 ± 4 AD-11237 aucgaaauauacugauccaTT 229uggaucaguauauuucgauTT 230  57 ± 3 AD-11238 ggaucccucgaaacuaacaTT 231uguuaguuucgagggauccTT 232  18 ± 1 AD-11239 acaccggacauuuauggugTT 233caccauaaauguccgguguTT 234  96 ± 2 AD-11240 guuuagugccacucgggccTT 235ggcccgaguggcacuaaacTT 236  95 ± 17 AD-11241 augacccgaacugaccuucTT 237gaaggucaguucgggucauTT 238  80 ± 7 AD-11242 auuuuagcgucauuacccuTT 239aggguaaugacgcuaaaauTT 240  70 ± 5 AD-11243 uuuuagcgucauuacccugTT 241caggguaaugacgcuaaaaTT 242  77 ± 8 AD-11244 gcaauagaucuccgugggaTT 243ucccacggagaucuauugcTT 244  23 ± 1 AD-11245 aaauaagcgaggcacuucuTT 245agaagugccucgcuuauuuTT 246  38 ± 3 AD-11246 auaagcgaggcacuucugaTT 247ucagaagugccucgcuuauTT 248  32 ± 2 AD-11247 ugauccuuacaaccagcgcTT 249gcgcugguuguaaggaucaTT 250  78 ± 2 AD-11248 uggccgagauaucucacucTT 251gagugagauaucucggccaTT 252  66 ± 1 AD-11249 caaccgccgcccacuagugTT 253cacuagugggcggcgguugTT 254  71 ± 4 AD-11250 uuagaugaaccuuuccgggTT 255cccggaaagguucaucuaaTT 256  90 ± 3 AD-11251 aaccuuuccgggcccaaagTT 257cuuugggcccggaaagguuTT 258  99 ± 7 AD-11252 auaaccuccguccuugaggTT 259ccucaaggacggagguuauTT 260 105 ± 2 AD-11253 cuugugacggaucccuuugTT 261caaagggauccgucacaagTT 262  62 ± 5 AD-11254 ccuaccuucgaagccaugcTT 263gcauggcuucgaagguaggTT 264  84 ± 6 AD-11255 aaaucauugccuucgacccTT 265gggucgaaggcaaugauuuTT 266  92 ± 6 AD-11256 cauugccuucgacccauacTT 267guaugggucgaaggcaaugTT 268  42 ± 2 AD-11257 cuucgacccauacuauuauTT 269auaauaguaugggucgaagTT 270  35 ± 2 AD-11258 ucaucgcuaauccgacuguTT 271acagucggauuagcgaugaTT 272  76 ± 2 AD-11259 aauccgacugugugggucuTT 273agacccacacagucggauuTT 274  87 ± 5 AD-11260 agcacgguggacugccuagTT 275cuaggcaguccaccgugcuTT 276  75 ± 7 AD-11261 ggugcgcaagacuugcuacTT 277guagcaagucuugcgcaccTT 278  34 ± 3 AD-11262 aggugcaucaacuauaccgTT 279cgguauaguugaugcaccuTT 280  59 ± 5 AD-11263 aucaacuauaccgauggagTT 281cuccaucgguauaguugauTT 282 113 ± 10 AD-11264 uugucgauugugaauaacaTT 283uguuauucacaaucgacaaTT 284  36 ± 5 AD-11265 aauggguuaccuugcacuuTT 285aagugcaagguaacccauuTT 286  70 ± 6 AD-11266 aggccuaucggagcuauguTT 287acauagcuccgauaggccuTT 288  72 ± 4 AD-11267 ggccuaucggagcuaugugTT 289cacauagcuccgauaggccTT 290  65 ± 7 AD-11268 uaucggagcuaugugcugcTT 291gcagcacauagcuccgauaTT 292 110 ± 10 AD-11269 ccguccuaugagagugucaTT 293ugacacucucauaggacggTT 294  50 ± 3 AD-11270 ccauuggaucccucgaaacTT 295guuucgagggauccaauggTT 296  18 ± 3 AD-11271 cauuggaucccucgaaacuTT 297aguuucgagggauccaaugTT 298  23 ± 3 AD-11272 aucccucgaaacuaacaacTT 299guuguuaguuucgagggauTT 300  76 ± 5 AD-11273 gaaacuaacaacuuccgucTT 301gacggaaguuguuaguuucTT 302  21 ± 3 AD-11274 uuccgucgcuuuacuccggTT 303ccggaguaaagcgacggaaTT 304  87 ± 6 AD-11275 ccgucgcuuuacuccggagTT 305cuccggaguaaagcgacggTT 306  29 ± 2 AD-11276 ggaucuagauccguucuacTT 307guagaacggaucuagauccTT 308  22 ± 3 AD-11277 cacacaccggacauuuaugTT 309cauaaauguccggugugugTT 310  57 ± 2 AD-11278 cacaccggacauuuaugguTT 311accauaaauguccggugugTT 312 101 ± 1 AD-11279 ggaccauuucccgguuuagTT 313cuaaaccgggaaaugguccTT 314  45 ± 4 AD-11280 accauuucccgguuuagugTT 315cacuaaaccgggaaaugguTT 316  89 ± 2 AD-11281 auuucccgguuuagugccaTT 317uggcacuaaaccgggaaauTT 318 100 ± 4 AD-11282 aaccugaucagaagaacggTT 319ccguucuucugaucagguuTT 320  66 ± 5 AD-11283 ucaguuuauuuauuacgguTT 321accguaauaaauaaacugaTT 322  76 ± 4 AD-11284 gugcaugacccgaacugacTT 323gucaguucgggucaugcacTT 324  29 ± 1 AD-11285 augaguucacguaccugagTT 325cucagguacgugaacucauTT 326  37 ± 4 AD-11286 agcgucauuacccuggcauTT 327augccaggguaaugacgcuTT 328  31 ± 1 AD-11287 gcgucauuacccuggcauaTT 329uaugccaggguaaugacgcTT 330  16 ± 1 AD-11288 gucauuacccuggcauaugTT 331cauaugccaggguaaugacIT 332  21 ± 1 AD-11289 acagcaauagaucuccgugTT 333cacggagaucuauugcuguTT 334  64 ± 4 AD-11290 ggacauucagaguucuuagTT 335cuaagaacucugaauguccTT 336  26 ± 1 AD-11291 ucuacauaaauaagcgaggTT 337ccucgcuuauuuauguagaTT 338  88 ± 15 AD-11292 uaaauaagcgaggcacuucTT 339gaagugccucgcuuauuuaTT 340  68 ± 2 AD-11293 ugcccugaugguuauaucuTT 341agauauaaccaucagggcaTT 342  39 ± 3 AD-11294 caacccggauuuuaacuacTT 343guaguuaaaauccggguugTT 344  26 ± 2 AD-11295 gggaaaaucuauaugaucuTT 345agaucauauagauuuucccTT 346  15 ± 1 AD-11296 gcccucgagaugcuccggaTT 347uccggagcaucucgagggcTT 348  54 ± 9 AD-11297 agggauugacacaaccucuTT 349agagguugugucaaucccuTT 350  22 ± 1 AD-11298 gggauugacacaaccucucTT 351gagagguugugucaaucccTT 352  22 ± 2 AD-11299 cacaauggaucaccuuuaaTT 353uuaaaggugauccauugugTT 354  23 ± 0 AD-11300 ccgcucugauccuuacaactT 355guuguaaggaucagagcggTT 356  34 ± 2 AD-11301 auccuuacaaccagcgcagTT 357cugcgcugguuguaaggauTT 358  90 ± 5 AD-11302 agcgcaggaugucuuuucuTT 359agaaaagacauccugcgcuTT 360  46 ± 3 AD-11303 cuuuucuaggccucgccucTT 361gaggcgaggccuagaaaagTT 362  94 ± 10 AD-11304 cuggaaaacgccgggcuagTT 363cuagcccggcguuuuccagTT 364  57 ± 2 AD-11305 aaacgccgggcuagucaugTT 365caugacuagcccggcguuuTT 366  95 ± 7 AD-11306 aacgccgggcuagucauggTT 367ccaugacuagcccggcguuTT 368 101 ± 5 AD-11307 agaccacgaaagccaucggTT 369ccgauggcuuucguggucuTT 370  89 ± 6 AD-11308 cucccuagaagcccucuucTT 371gaagagggcuucuagggagTT 372  50 ± 3 AD-11309 agaugaacaccaaccgccgTT 373cggcgguugguguucaucuTT 374  62 ± 7 AD-11310 augaacaccaaccgccgccTT 375ggcggcgguugguguucauTT 376  99 ± 6 AD-11311 aaccgccgcccacuagugaTT 377ucacuagugggcggcgguuTT 378  72 ± 2 AD-11312 accgccgcccacuagugagTT 379cucacuagugggcggcgguTT 380  99 ± 13 AD-11313 gcugucgaugucucggcauTT 381augccgagacaucgacagcTT 382  67 ± 8 AD-11314 ucgaugucucggcauucgaTT 383ucgaaugccgagacaucgaTT 384  36 ± 2 AD-11315 ucucggcauucgaugcaggTT 385ccugcaucgaaugccgagaTT 386  99 ± 5 AD-11316 ggcauucgaugcaggacaaTT 387uuguccugcaucgaaugccTT 388  48 ± 5 AD-11317 gcauucgaugcaggacaaaTT 389uuuguccugcaucgaaugcTT 390  56 ± 5 AD-11318 cuuagaugaaccuuuccggTT 391ccggaaagguucaucuaagTT 392  47 ± 3 AD-11319 gaguguugucaguaucauaTT 393uaugauacugacaacacucTT 394  22 ± 2 AD-11320 caguaucauaaccuccgucTT 395gacggagguuaugauacugTT 396  27 ± 2 AD-11321 cucgaggagucugaacagaTT 397ucuguucagacuccucgagTT 398  32 ± 2 AD-11322 guaucugaucugggauugcTT 399gcaaucccagaucagauacTT 400  65 ± 3 AD-11323 agacaauucucuuugggcuTT 401agcccaaagagaauugucuTT 402  73 ± 2 AD-11324 caccuugugcaucguggugTT 403caccacgaugcacaaggugTT 404  47 ± 4 AD-11325 gcaugagcccuaccuucgaTT 405ucgaagguagggcucaugcTT 406  25 ± 1 AD-11326 uaccuucgaagccaugcucTT 407gagcauggcuucgaagguaTT 408  71 ± 2 AD-11327 caaaaucauugccuucgacTT 409gucgaaggcaaugauuuugTT 410  33 ± 3 AD-11328 aucauugccuucgacccauTT 411augggucgaaggcaaugauTT 412  38 ± 2 AD-11329 ucauugccuucgacccauaTT 413uaugggucgaaggcaaugaTT 414  29 ± 1 AD-11330 auugccuucgacccauacuTT 415aguaugggucgaaggcaauTT 416  77 ± 7 AD-11331 ucgacccauacuauuauuuTT 417aaauaauaguaugggucgaTT 418  55 ± 3 AD-11332 caucaucgucacugugaguTT 419acucacagugacgaugaugTT 420  31 ± 3 AD-11333 caucgucacugugagucugTT 421cagacucacagugacgaugTT 422  33 ± 4 AD-11334 ucgucacugugagucugcuTT 423agcagacucacagugacgaTT 424  44 ± 4 AD-11335 ggaaaacuaccguaacaacTT 425guuguuacgguaguuuuccTT 426  34 ± 1 AD-11336 uaccguaacaaccgaaaaaTT 427uuuuucgguuguuacgguaTT 428  21 ± 3 AD-11337 cguaacaaccgaaaaaauaTT 429uauuuuuucgguuguuacgTT 430  29 ± 2 AD-11338 aaaauccauaugccucaucTT 431gaugaggcauauggauuuuTT 432  87 ± 4 AD-11339 cuguucaucgcccugcuauTT 433auagcagggcgaugaacagTT 434  40 ± 2 AD-11340 uguucaucgcccugcuauuTT 435aauagcagggcgaugaacaTT 436  26 ± 1 AD-11341 cccugcuauugaacucuuuTT 437aaagaguucaauagcagggTT 438  29 ± 1 AD-11342 ggccaucguaccaaacaggTT 439ccuguuugguacgauggccTT 440  41 ± 2 AD-11343 ccaucguaccaaacaggcuTT 441agccuguuugguacgauggTT 442  47 ± 2 AD-11344 cagugacuucaucgcuaauTT 443auuagcgaugaagucacugTT 444  33 ± 2 AD-11345 cuucaucgcuaauccgacuTT 445agucggauuagcgaugaagTT 446  36 ± 2 AD-11346 uaauccgacugugugggucTT 447gacccacacagucggauuaTT 448  97 ± 3 AD-11347 gaaucugaucuugaugacuTT 449agucaucaagaucagauucTT 450  45 ± 2 AD-11348 aagaguccaugggauguggTT 451ccacaucccauggacucuuTT 452 100 ± 2 AD-11349 gcaggugcgcaagacuugcTT 453gcaagucuugcgcaccugcTT 454  39 ± 3 AD-11350 gcgcaagacuugcuaccguTT 455acgguagcaagucuugcgcTT 456  29 ± 1 AD-11351 aagacuugcuaccguaucgTT 457cgauacgguagcaagucuuTT 458  82 ± 2 AD-11352 gacuugcuaccguaucgugTT 459cacgauacgguagcaagucTT 460  56 ± 2 AD-11353 cucauugugaauaucucacTT 461gugagauauucacaaugagTT 462  54 ± 3 AD-11354 ugauaagucucacagcgaaTT 463uucgcugugagacuuaucaTT 464  31 ± 1 AD-11355 aucaaagcccuucgaacccTT 465ggguucgaagggcuuugauTT 466  87 ± 8 AD-11356 aaagcccuucgaacccuucTT 467gaaggguucgaagggcuuuTT 468  83 ± 3 AD-11357 ccuucgaacccuucgcgcuTT 469agcgcgaaggguucgaaggTT 470  50 ± 1 AD-11358 cuucgaacccuucgcgcucTT 471gagcgcgaaggguucgaagTT 472  55 ± 4 AD-11359 ugcaucaacuauaccgaugTT 473caucgguauaguugaugcaTT 474  59 ± 7 AD-11360 cccuuguaccuuugucgauTT 475aucgacaaagguacaagggTT 476  24 ± 3 AD-11361 uuguaccuuugucgauuguTT 477acaaucgacaaagguacaaTT 478  39 ± 6 AD-11362 uuugauaauguugcaauggTT 479ccauugcaacauuaucaaaTT 480 110 ± 14 AD-11363 gcaauggguuaccuugcacTT 481gugcaagguaacccauugcTT 482  93 ± 6 AD-11364 uggguuaccuugcacuucuTT 483agaagugcaagguaacccaTT 484  54 ± 11 AD-11365 gcuguugauucccgggaggTT 485ccucccgggaaucaacagcTT 486  36 ± 1 AD-11366 aucgugaccagacaagcuuTT 487aagcuugucuggucacgauTT 488  36 ± 5 AD-11367 cgucuucacaggcgaauguTT 489acauucgccugugaagacgTT 490  57 ± 2 AD-11368 ucacaggcgaaugugucauTT 491augacacauucgccugugaTT 492  76 ± 5 AD-11369 cacaggcgaaugugucaugTT 493caugacacauucgccugugTT 494  75 ± 4 AD-11370 caggcgaaugugucaugaaTT 495uucaugacacauucgccugTT 496  32 ± 5 AD-11371 aggcgaaugugucaugaagTT 497cuucaugacacauucgccuTT 498  48 ± 6 AD-11372 uguucgcuuugaggcaguaTT 499uacugccucaaagcgaacaTT 500  36 ± 4 AD-11373 gcaguacuacuucacaaauTT 501auuugugaaguaguacugcTT 502  27 ± 2 AD-11374 uccauugcgagccugauuuTT 503aaaucaggcucgcaauggaTT 504  24 ± 2 AD-11375 ccauugcgagccugauuuuTT 505aaaaucaggcucgcaauggTT 506  21 ± 3 AD-11376 aucgggcuguugcuauuccTT 507ggaauagcaacagcccgauTT 508  78 ± 11 AD-11377 aaaacccaaucgaaauauaTT 509uauauuucgauuggguuuuTT 510  33 ± 4 AD-11378 caaucgaaauauacugaucTT 511gaucaguauauuucgauugTT 512  66 ± 8 AD-11379 aaucgaaauauacugauccTT 513ggaucaguauauuucgauuTT 514 128 ± 8 AD-11380 ucgaaauauacugauccagTT 515cuggaucaguauauuucgaTT 516 119 ± 7 AD-11381 aaucauccuaugaaccaauTT 517auugguucauaggaugauuTT 518  39 ± 3 AD-11382 uaugaaccaauagcaaccaTT 519ugguugcuauugguucauaTT 520  61 ± 4 AD-11383 cacucuccgauggaagcaaTT 521uugcuuccaucggagagugTT 522  69 ± 5 AD-11384 cuaucggagcuaugugcugTT 523cagcacauagcuccgauagTT 524  95 ± 10 AD-11385 agcaaaugaaaauuguguaTT 525uacacaauuuucauuugcuTT 526  79 ± 6 AD-11386 uacucccagacaaaucugaTT 527ucagauuugucugggaguaTT 528  36 ± 4 AD-11387 agagugucacuagaggccuTT 529aggccucuagugacacucuTT 530  36 ± 1 AD-11388 ggccuuagugauagagucaTT 531ugacucuaucacuaaggccTT 532  35 ± 4 AD-11389 agugauagagucaacaugaTT 533ucauguugacucuaucacuTT 534  29 ± 5 AD-11390 gauagagucaacaugaggaTT 535uccucauguugacucuaucTT 536  32 ± 3 AD-11391 caucuagcucaauacaaaaTT 537uuuuguauugagcuagaugTT 538  23 ± 3 AD-11392 cuagcucaauacaaaaugaTT 539ucauuuuguauugagcuagTT 540  28 ± 5 AD-11393 aguauggagcugauugcccTT 541gggcaaucagcuccauacuTT 542 108 ± 8

Example 2 Optimization of siRNAs by Chemical Modification

As has been experienced by those working in the antisense field,ribonucleic acids are often quickly degraded by a range of nucleasespresent in virtually all biological environments, e.g. endonucleases,exonucleases etc. This vulnerability may be circumvented by chemicallymodifying these oligonucleotides such that nucleases may no longerattack. Consequently, siRNAs in Table 1 represent chemically modifiedoligonucleotides; these chemically modified siRNAs were tested forinhibitory activity on Nav1.8 gene expression (Nav1.8 mRNA levels).

dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as −Chol or −sChol, depending on whether the link to the cholesterylgroup is effected via a phosphodiester or a phosphorothioate diestergroup), an appropriately modified solid support was used for RNAsynthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature untilcompletion of the reaction was ascertained by TLC. After 19 h thesolution was partitioned with dichloromethane (3×100 mL). The organiclayer was dried with anhydrous sodium sulfate, filtered and evaporated.The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.Completion of the reaction was ascertained by TLC. The reaction mixturewas concentrated under vacuum and ethyl acetate was added to precipitatediisopropyl urea. The suspension was filtered. The filtrate was washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer was dried over sodium sulfate and concentrated togive the crude product which was purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl esterAB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated under vacuum, water was addedto the residue, and the product was extracted with ethyl acetate. Thecrude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[α]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 mL of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water Theresultant mixture was extracted twice with 100 mL of dichloromethaneeach and the combined organic extracts were washed twice with 10 mL ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 mL of toluene, cooled to 0° C. and extracted with three50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40mL portions of chloroform which were combined, dried and evaporated todryness. The residue was purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) was added, the mixture was extracted with ethylacetate (3×40mL). The combined ethylacetate layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out at room temperature overnight.The reaction was quenched by the addition of methanol. The reactionmixture was concentrated under vacuum and to the residue dichloromethane(50 mL) was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The achieved loading of the CPG was measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) was performed as described inWO 2004/065601, except that, for the cholesteryl derivative, theoxidation step was performed using the Beaucage reagent in order tointroduce a phosphorothioate linkage at the 5′-end of the nucleic acidoligomer.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 2.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation^(a) Nucleotide(s) A, a2′-deoxy-adenosine-5′-phosphate, adenosine-5′-phosphate C, c2′-deoxy-cytidine-5′-phosphate, cytidine-5′-phosphate G, g2′-deoxy-guanosine-5′-phosphate, guanosine-5′-phosphate T, t2′-deoxy-thymidine-5′-phosphate, thymidine-5′-phosphate U, u2′-deoxy-uridine-5′-phosphate, uridine-5′-phosphate N, n any2′-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c or u) Am2′-O-methyladenosine-5′-phosphate Cm 2′-O-methylcytidine-5′-phosphate Gm2′-O-methylguanosine-5′-phosphate Tm 2′-O-methyl-thymidine-5′-phosphateUm 2'-O-methyluridine-5'-phosphate Af2′-fluoro-2′-deoxy-adenosine-5′-phosphate Cf2′-fluoro-2′-deoxy-cytidine-5′-phosphate Gf2′-fluoro-2′-deoxy-guanosine-5′-phosphate Tf2′-fluoro-2′-deoxy-thymidine-5′-phosphate Uf2′-fluoro-2′-deoxy-uridine-5′-phosphate A, C, G, T, U, a, underlined:nucleoside-5′-phosphorothioate c, g, t, u am, cm, gm, tm, underlined:2-O-methyl-nucleoside-5′-phosphorothioate um ^(a)capital lettersrepresent 2′-deoxyribonucleotides (DNA), lower case letters representribonucleotides (RNA)

Single-Dose Screen of Nav1.8 siRNAs Against mRNA Expression ofTransfected Human Nav1.8 in Cos-7 Cells.

All Nav1.8 siRNAs in Table 1 and Table 4 were tested initially at asingle dose of 100 nM (Table 1) or 50 nM (Table 4) for activity inreducing mRNA expression of transfected human Nav1.8 in Cos-7 cells. Oneday before transfection, Cos-7 cells (DSMZ, Braunschweig, Germany) wereseeded at 1.5×10⁴ cells/well on 96-well plates (Greiner Bio-One GmbH,Frickenhausen, Germany) in 100 μl of growth medium (Dulbecco's MEM, 10%fetal calf serum, 2 mM L-glutamine, 1.2 μg/ml sodium bicarbonate, 100upenicillin/100 μg/ml streptomycin, all from Biochrom AG, Berlin,Germany). Four hours prior to siRNA transfection, 20 ng of plasmid/well(Table 1) or 50 ng/well (Table 4) were transfected withLipofectamine-2000 (Invitrogen) as described below for the siRNAs, withthe plasmid diluted in Opti-MEM to a final volume of 12.5 μl/well,prepared as a mastermix for the whole plate.

siRNA transfections were performed in triplicate. For each well, 0.5 μlLipofectamine-2000 (Invitrogen GmbH, Karlsruhe, Germany) was mixed with12 μl Opti-MEM (Invitrogen) and incubated for 15 min at roomtemperature. For an siRNA concentration of 100 nM in a transfectionvolume of 100 μl, 2 μl of a 5 μM siRNA were mixed with 10.5 μl Opti-MEMper well, combined with the Lipofectamine-2000-Opti-MEM mixture andagain incubated for 15 minutes at room temperature. During thatincubation time, growth medium was removed from cells and replaced by 75μl/well of fresh medium. In six wells, growth medium was replaced by 100μl of fresh medium and cells were lysed immediately by adding lysismixture, as described below, in order to analyse the background value inthe bDNA-assay caused by the Nav1.8-cDNA in the plasmid.siRNA-Lipofectamine-2000-complexes were applied completely (25 μl eachper well) to the cells and cells were incubated for 24 h at 37° C. and5% CO₂ in a humidified incubator (Heraeus GmbH, Hanau, Germany).

Cells were harvested by applying 50 μl of lysis mixture (from theQuantiGene bDNA-kit from Genospectra, Fremont, USA) to each well andwere lysed at 53° C. for 30 min. Afterwards, 50 μl of the lysates wereincubated with probesets specific to human Nav1.8 and human GAPDH(sequence of probesets see below) and processed according to themanufacturer's protocol for QuantiGene. Chemoluminescence was measuredin a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relativelight units) and values obtained with the human Nav1.8 probeset werenormalized to the respective human GAPDH (GAPDH sequence ofCercopithecus aethiops so far unknown) values for each well. Valuesobtained with cells lysed 4 h after plasmid transfection were subtractedfrom the values obtained with cells lysed 24 h after siRNA transfection.Values acquired with siRNAs directed against Nav1.8 were furthernormalized relative to the value obtained with an unrelated siRNA(directed against hepatitis C virus) which was set to 100%.

FIG. 1 provides the results from a representative experiment wheresiRNAs from Table 1 were tested at a single dose of 100 nM and Table 4provides the results for additional siRNAs at a dose of 50 nM. SeveralsiRNAs (in Tables 1 and 4) were effective at the dose tested in reducingNav1.8 mRNA levels by at least 50% in COS-7 cells transfected withNav1.8.

Dose-Response Curves for Selected siRNAs Against mRNA Expression ofTransfected Human Nav1.8 in Cos-7 Cells

Several effective siRNAs against Nav1.8 from the single dose screen(results in FIG. 1) were further characterized by dose response curves.For dose response curves, transfections were performed as for the singledose screen above, but with the following concentrations of siRNA (nM):100, 33, 11, 3.7, 1.2, 0.4, 1, 0.14, 0.05, 0.015, 0.005 and mock (nosiRNA). siRNAs were diluted with Opti-MEM to a final volume of 12.5 μlaccording to the above protocol.

Three independent dose response experiments were carried out to generatedose response curves (DRCs). The dose response curves were repeated, anda summary of the results are provided in Table 3.

TABLE 3 IC50 values for selected siRNAs targeting Nav1.8 IC50-values[nM]: 1st DRC screen 2nd DRC screen AL-DP-6218 0.0068 0.021 AL-DP-62170.036 0.0021 AL-DP-6050 0.013 0.16 AL-DP-6209 0.060 0.012 AL-DP-6042 nd0.016 AL-DP-6049 0.24 0.0010 AL-DP-6219 0.13 0.0069 AL-DP-6223 73140 22AL-DP-6225 0.70 8.07

Specificity Testing of the Nav1.8 siRNAs Against Nav1.5 mRNA Nav1.5 isan NaV subtype that is closely related to Nav1.8, but has an importantrole in normal cardiac function. Therefore, it is important to confirmthat siRNAs selective for Nav1.8 do not inhibit Nav1.5 expression.Several siRNAs were tested for specificity towards Nav1.8 bytransfecting SW620 cells, which express endogenous human Nav1.5, withthese siRNAs, and assessing Nav1.5 mRNA levels. A control unrelatedsiRNA (AL-DP-5002) was also transfected into SW620 cells. siRNAstargeting Nav1.8 were tested at the following doses: 1200 nM, 400 nM,133.3 nM, 44.4 nM, 14.8 nM, 4.9 nM, 1.6 nM, and mock (without siRNA).One siRNA targeting Nav1.8 (AL-DP-6217) was tested at 1 nM, 10 nM, 100nM and 1 uM. The expression of Nav1.5 mRNA, which encodes the proteinwith the highest homology to Nav1.8 in the Nav-family, was thenquantified.

One day before transfection, SW620 cells (LCG Promochem, Wesel, Germany)were seeded at 1.5×10⁴ cells/well on 96-well plates (Greiner Bio-OneGmbH, Frickenhausen, Germany) in 100 μl of growth medium (Leibowitz L-15Medium, 10% fetal calf serum, 2 mM L-glutamine, 100 u penicillin/100μg/ml streptomycin, all from Biochrom AG, Berlin, Germany).

siRNA transfections were performed in triplicate. For each well, 0.6 μlOligofectamine (Invitrogen GmbH, Karlsruhe, Germany) was mixed with 2.4μl Opti-MEM (Invitrogen) and incubated for 10 min at room temperature.For an siRNA concentration of 1200 nM in 100 μl transfection volume, 5μl of 24 μM siRNA was mixed with 12 μl Opti-MEM per well, combined withthe Oligofectamine-Opti-MEM mixture and again incubated for 20 minutesat room temperature. During that incubation time, growth medium wasremoved from cells and replaced by 80 μl/well of fresh growth mediumwithout serum. siRNA-Oligofectamine-complexes were applied completely(20 μl each per well) to the cells and cells were incubated for 24 h at37° C. without CO₂ in a humidified incubator (Heraeus GmbH, Hanau,Germany).

Cells were harvested by applying 50 μl of lysis mixture (content of theQuantiGene bDNA-kit from Genospectra, Fremont, USA) to each well andwere lysed at 53° C. for 30 min. Afterwards, 50 μl of the lysates wereincubated with probesets specific to human Nav1.5 and human GAPDH(sequence of probesets see below) and processed according to themanufacturer's protocol for QuantiGene. Chemoluminescence was measuredin a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relativelight units) and values obtained with the human Nav1.5 probeset werenormalized to the respective human GAPDH values for each well. Anunrelated control siRNA (directed against hepatitis C virus) was used asa negative control.

FIG. 2 provides the results. At the concentrations tested, the selectedsiRNAs targeting Nav1.8, did not exhibit significant dose-dependentinhibition of Nav1.5 mRNA as compared with the unrelated control siRNA(AL-DP-5002), confirming the specificity of these Nav1.8 siRNAs forNav10.8 over Nav10.5.

In addition, at concentrations up to 1 uM, AL-DP-6217 exhibited nosignificant inhibition of Nav1.5 mRNA (data not shown), confirming thespecificity of this Nav1.8 siRNA for Nav1.8 over Nav1.5.

TABLE 5 bDNA Probesets for the quantitation of human Nav1.8, Nav1.5 andGAPDH Human Nav1.8 probeset: SEQ ID FPL Name Function Sequence NO:hNa182001 CE TTTGGAGGTTAAAGGTGATCCATTTTTTCTCTTGGAAAGAAAGT 543 hNa182002CE GGCGTTTTCCAGAGGCGAGTTTTTCTCTTGGAAAGAAAGT 544 hNa182003 CECCAGCAGCAGAGAGCCCCTTTTTCTCTTGGAAAGAAAGT 545 hNa182004 CECTGGGTTGAGGAAGAGGGCTTTTTTCTCTTGGAAAGAAAGT 546 hNa182005 CEAACACTCATTGCCCTTTGGGTTTTTCTCTTGGAAAGAAAGT 547 hNa182006 CEAAGGACGGAGGTTATGATACTGACTTTTTCTCTTGGAAAGAAAGT 548 hNa182007 CETGTTCAGACTCCTCGAGTTCCTCTTTTTCTCTTGGAAAGAAAGT 549 hNa182008 LECTATGCCTTCTCTCACTGGCATTTTTTTAGGCATAGGACCCGTGTCT 550 hNa182009 LECTGGTTGTAAGGATCAGAGCGGTTTTTAGGCATAGGACCCGTGTCT 551 hNa182010 LEAACACACTGCCATGACTAGCCCTTTTTAGGCATAGGACCCGTGTCT 552 hNa182011 LEGATGGCTTTCGTGGTCTCCATTTTTAGGCATAGGACCCGTGTCT 553 hNa182012 LECTGGCCAGCACCCCCACTTTTTAGGCATAGGACCCGTGTCT 554 hNa182013 LECATGCCTGGAGTCAGGGTTGTTTTTAGGCATAGGACCCGTGTCT 555 hNa182014 LEAGACATCGACAGCTCCAGGGTTTTTAGGCATAGGACCCGTGTCT 556 hNa182015 LEGTCCTGCATCGAATGCCGTTTTTAGGCATAGGACCCGTGTCT 557 hNa182016 LECAAGCAGGGTGGGCACTTCTTTTTAGGCATAGGACCCGTGTCT 558 hNa182017 BLTGTGGGAGTGGAGAGAGGTTG 559 hNa182018 BL CCCTCTGACACTCTTGGCTTTATT 560hNa182019 BL GGTGATTTGTTGTCTTCTGTGGAG 561 hNa182020 BLGCCTAGAAAAGACATCCTGCG 562 hNa182021 BL GCCAGGGGACCGGAAATGG 563 hNa182022BL CCCTCAGGGAGTGAGATATCTCG 564 hNa182023 BL GGAAAGACTCCATCATCTGTGACT 565hNa182024 BL TCTAGGGAGGGGGCCTTG 566 hNa182025 BL GCGGTTGGTGTTCATCTTCTC567 hNa182026 BL GCAAGCTCACTAGTGGGCG 568 hNa182027 BLTCTGCTGACAAGAAAGTCTTCTTTT 569 hNa182028 BL CCCGGAAAGGTTCATCTAAGTAT 570Human GAPDH probeset: SEQ ID FPL Name Function Sequence NO: hGAP001 CEGAATTTGCCATGGGTGGAATTTTTTCTCTTGGAAAGAAAGT 571 hGAP002 CEGGAGGGATCTCGCTCCTGGATTTTTCTCTTGGAAAGAAAGT 572 hGAP003 CECCCCAGCCTTCTCCATGGTTTTTTCTCTTGGAAAGAAAGT 573 hGAP004 CEGCTCCCCCCTGCAAATGAGTTTTTCTCTTGGAAAGAAAGT 574 hGAP005 LEAGCCTTGACGGTGCCATGTTTTTAGGCATAGGACCCGTGTCT 575 hGAP006 LEGATGACAAGCTTCCCGTTCTCTTTTTAGGCATAGGACCCGTGTCT 576 hGAP007 LEAGATGGTGATGGGATTTCCATTTTTTTAGGCATAGGACCCGTGTCT 577 hGAP008 LEGCATCGCCCCACTTGATTTTTTTTTAGGCATAGGACCCGTGTCT 578 hGAP009 LECACGACGTACTCAGCGCCATTTTTAGGCATAGGACCCGTGTCT 579 hGAP010 LEGGCAGAGATGATGACCCTTTTGTTTTTAGGCATAGGACCCGTGTCT 580 hGAP011 BLGGTGAAGACGCCAGTGGACTC 581 Human Nav1.5 probeset: SEQ ID FPL NameFunction Sequence NO: hNa15001 CECGTTTTCTCCTCTTGCTTCTTCTCTTTTTCTCTTGGAAAGAAAGT 582 hNa15002 CEGGGAGCCTGTCCTCCCCATTTTTCTCTTGGAAAGAAAGT 583 hNaI5003 CETCGCCTGCGAAAGGTGAATTTTTCTCTTGGAAAGAAAGT 584 hNaIS004 CECTCTCGCTCTCCCCCGCTTTTTCTCTTGGAAAGAAAGT 585 hNa15005 CECCGGGACTGGGCTGTCCTTTTTCTCTTGGAAAGAAAGT 586 hNa15006 CETTTTGCCATGGAGGGCGTTTTTTCTCTTGGAAAGAAAGT 587 hNa15007 LEGGCTGAGATGATTCATTGCTCTGTTTTTAGGCATAGGACCCGTGTCT 588 hNa15008 LEGCTGAGGCCACGGGTGATTTTTAGGCATAGGACCCGTGTCT 589 hNa15009 LEAATGCTCCCGCGGCTGGTTTTTAGGCATAGGACCCGTGTCT 590 hNa15010 LECTGGGCACTGGTCCGGCTTTITTAGGCATAGGACCCGTGTCT 591 hNa15011 LEGGCCAGGAGCCGAGGTTTTTTTAGGCATAGGACCCGTGTCT 592 hNa15012 LECCCAGTAATGAGACCACCCCATTTTTTAGGCATAGGACCCGTGTCT 593 hNa15013 LECCTCTGGGTCGCCTGCCTTTTTAGGCATAGGACCCGTGTCT 594 hNa15014 BLCACTCCTCAGTTCCTGAAGACATC 595 hNa15015 BL GGACCATCTTCTGAGTCAGACTTG 596hNa15016 BL AACGTGGCTTCATAGAAGTCCT 597 hNa15017 BLAATCTGCTTCAGAACCCAGGTC 598 hNa15018 BL TGTGCTGTTTTCATCATCTGCAA 599hNa15019 BL CCAGCAGTGATGTGTGGTGG 600 hNa15020 BL GCAGGGGCCAGGGCA 601hNa15021 BL TGCAGTCCACAGTGCTGTTCT 602 hNa15022 BL GGCTTCCTGGGGATGTGG 603

Single-Dose Screen of Nav1.8 siRNAs Against mRNA Expression ofEndogenous Nav1.8 in Primary Cultures of Rat Dorsal Root Ganglion Cells.

To confirm and extend the results obtained on mRNA expression oftransfected human Nav1.8 in Cos-7 cells, Nav1.8 siRNAs from Table 1 weretested at a single dose of 200 nM for activity in reducing mRNAexpression of endogenous Nav1.8 in primary cultures of rat dorsal rootganglion (DRG) cells.

DRG cells were isolated from Sprague-Dawley rats at postnatal day 3 to6. DRGs were dissected and cells dissociated into single cells byincubation with 0.28 Wunsch units/ml Liberase Blendzyme (Roche) in S-MEM(Gibco) at 37° C. for 35 min. The cell suspension was pre-plated ontissue-culture plates to remove non-neuronal cells. Neurons were thenplated onto tissue-culture Biocoat™ PDL Poly-D-Lysine/Laminin 96-wellplates (BD Biosciences, Bedford Mass., USA) in F12-HAM's Mediumcontaining glutamine (Invitrogen Gibco, Carlsbad Calif., USA) with 5%fetal bovine serum (FBS, heat inactivated) and 5% horse serum (heatinactivated) (both Invitrogen Gibco, Carlsbad Calif., USA) supplementedwith 50 ng/ml mouse nerve growth factor 2.5S (NGF; Promega Corp.,Madison Wis., USA) and kept at 37° C., 5% CO₂ in a humidified incubatoruntil transfection.

Nav1.8 siRNAs were screened in DRG cultures at 200 nM in duplicate usingTransMessenger™ Transfection reagent (Qiagen GmbH, Hilden, Germany, cat.no. 301525) which is based on a lipid formulation, a specificRNA-condensing reagent (Enhancer R™) and an RNA-condensing buffer(Buffer EC-R™), keeping siRNA:Enhancer R™ ratio (μg:μl) constant at 1:2,and siRNA:TransMessenger™ ratio (μg:μl) constant at 1:12.

DRG neurons were transfected 24 h post-plating. For each well, 0.52 μlEnhancer R™ were first mixed with 13.68 μl Buffer EC-R™. 0.8 μl of a 25μM solution of AL-DP-5987 (0.26 μg) in annealing buffer (20 mM sodiumphosphate, pH 6.8; 100 mM sodium chloride), or 0.8 μl of annealingbuffer (siRNA-free control) were added and the mixture incubated for 5min at RT. 3.12 μl TransMesssenger™ Transfection Reagent were dilutedwith 6.88 μl Buffer EC-R added to the mixture, and the mixture incubatedfor another 10 min at room temperature to allow transfection-complexformation. 75 μl serum free F12-HAM's Medium containing glutamine(Invitrogen Gibco, Carlsbad Calif., USA) supplemented with 50 ng/ml NGF2.5S (Promega Corp., Madison Wis., USA) and 1:50 B27 supplement(Invitrogen Gibco, Carlsbad Calif., USA) were added to the transfectioncomplexes and complete mixing achieved by gently pipetting up and down.The growth medium was removed from the DRG cells, and 90 μl of the abovetransfection complex mixture were added onto the cells. After 7 to 8 hof incubation at 37° C., 5% CO₂ in a humidified incubator supernatantwas removed from the cells, fresh F12-HAM's medium containing glutaminesupplemented with 5% FBS, 5% horse serum (both Invitrogen Gibco,Carlsbad Calif., USA), 50 ng/ml mouse NGF 2.5S (Promega Corp., MadisonWis., USA) and 1:100 Penicillin/Streptomycin (Invitrogen Gibco, CarlsbadCalif., USA) was added, the cells were incubated for another 16 h at 37°C., 5% CO₂ in a humidified incubator, and Nav1.8 mRNA was quantified.

Nav1.8 mRNA levels were measured using the QuantiGene™ bDNA kit(Genospectra, Fremont, USA) according to manufacturer's protocol.Briefly, the supernatant was removed from the DRG cells, and the cellswere lysed by addition of 150 μl of Lysis Working Reagent (I volume ofLysis Mixture plus 2 volumes of medium) and incubation at 52° C. for 30min. 40 μl of the lysates were incubated at 52° C. for 40 min with theprobe sets specific to rat Nav1.8 and mouse synuclein (SNCL).Chemoluminescence was read on a Victor²-Light™ (PerkinElmer Life AndAnalytical Sciences, Inc., Boston Mass., USA) as Relative Light Units(RLU). RLU for Nav1.8 were normalized to SNCL RLU for each well.Normalized Nav1.8/SNCL ratios were then compared to the siRNA-freecontrol, which was set as 100%.

FIG. 3 provides the results. At 200 nM, at least 13 (indicated by ‘*’)of the Nav1.8 siRNAs tested showed at least 50% Nav1.8 mRNA knock downcompared to the siRNA-free control (TransMessenger™ only; TM only),while an unrelated control siRNA against RhoA had no effect.

Dose Response of dsRNA AL-DP-6209 Against mRNA Expression of EndogenousNav1.8 in Primary Cultures of Rat Dorsal Root Ganalion Cells

One effective siRNA (AL-DP-6209) against Nav1.8 from the single dosescreen in primary cultures of rat dorsal root ganglion cells was furthercharacterized for dose dependence. For the dose response curve,experiments were performed as for the single dose screen in DRG culturesabove, but with the following concentrations of siRNA: 175, 88, 44, 22,11 and 5.5 nM. For all siRNA concentrations, siRNA:Enhancer R ratio(ug:ul) was kept constant at 1:2 and siRNA:TransMessenger ratio (ug/ul)was kept constant at 1:12 (FIG. 4).

FIG. 4 provides the result for the selected siRNA AL-DP-6209 from a doseresponse experiment. At 5.5 and 11 nM, AL-DP-6209 did not inhibit Nav1.8mRNA expression relative to SNCL, whereas at 88 and 175 nM, AL-DP-6209inhibited Nav1.8 mRNA expression relative to SNCL by >40%. Maximalinhibition of Nav1.8 mRNA expression relative to SNCL occurred in thisexperiment at 175 nM.

Intrathecal Bolus Administration of siRNAs Against Nav1.8 with iFECTPrevents Inflammatory Pain

The effect of siRNAs against Nav1.8, formulated with iFECT, on completeFreund's adjuvant-induced tactile hypersensitivity was evaluated in rats(FIG. 5). Adult male Sprague-Dawley rats received an injection of CFA(150 uL) into the hindpaw on day 0. siRNAs against Nav1.8 were thenadministered by intrathecal bolus to the lumbar region of the spinalcord on days 1, 2 and 3; specifically, for each bolus injection, 2 ug ofsiRNA was complexed with iFECT transfection reagent (Neuromics,Minneapolis Minn., USA) at a ratio of 1:4 (w:v) in a total volume of 10uL. Five groups of rats (with 5 rats per group) were treated with eithersiRNA (AL-DP-6049, AL-DP-6209, AL-DP-6217 or AL-DP-6218; Table 1), orPBS, in the presence of iFECT. Tactile hypersensitivity was expressed astactile withdrawal thresholds which were measured by probing the hindpawwith 8 calibrated von Frey filaments (Stoelting, Wood Dale Ill., USA)(0.41 g to 15 g). Each filament was applied to the plantar surface ofthe paw. Withdrawal threshold was determined by sequentially increasingand decreasing the stimulus strength and calculated with a Dixonnon-parametric test (see Dixon, W. J. (1980) “Efficient analysis ofexperimental observations” Annu Rev Pharmacol Toxicol 20:441-462;Chaplan, S. R., F. W. Bach, et al. (1994) “Quantitative assessment oftactile allodynia in the rat paw” J Neurosci Methods 53:55-63). Tactilethresholds were measured before CFA injection to assess baselinethresholds, and then on day 4 after CFA and treatment with testarticles. In rats treated with PBS, tactile hypersensitivity waspronounced on day 4, as evidenced by reduced paw withdrawal threshold,as expected. In rats treated with AL-DP-6209, tactile thresholds werenearly normalized on day 4, demonstrating that the Nav1.8 siRNA,AL-DP-6209, is efficacious in vivo against inflammation-inducedhyperalgesia. Treatment with the Nav1.8 siRNA, AL-DP-6217, resulted inthe average tactile threshold trending towards baseline, with one offive rats demonstrating a normal tactile response. AL-DP-6049 andAL-DP-6218 did not significantly alter tactile thresholds compared toPBS treatment, in this experimental paradigm.

These results demonstrate that siRNAs targeting Nav1.8, formulated withtransfection reagent and administered intrathecally, alleviateCFA-induced tactile hyperalgesia, and therefore represent a novelapproach to providing effective treatment of clinical inflammatory pain.

Intrathecal Bolus Administration of siRNAs Against Nav1.8 withoutTransfection Reagent Alleviates Inflammatory Pain

The effect of siRNAs against Nav1.8, formulated in phosphate bufferedsaline (PBS), on complete Freund's adjuvant (CFA)-induced tactilehypersensitivity was evaluated in rats (FIG. 6). Of 3 Nav1.8 siRNAstested, 2 siRNAs were efficacious against CFA-induced tactilehypersensitivity: AL-DP-4461, an unconjugated siRNA with differentchemical modifications but the same sequence as AL-DP-6050, andAL-DP-4459, a cholesterol-conjugated siRNA with the same chemicalmodifications and sequence as AL-DP-6050. With the dosing paradigmsevaluated in this experiment, AL-DP-6980 (cholesterol-conjugated siRNAwith the same chemical modifications and sequence as AL-DP-6209) was notefficacious against CFA-induced tactile hypersensitivity. The sequencesof AL-DP-4461, AL-DP-4459 and AL-DP-6980 are shown in Table 6.

TABLE 6 Further modified siRNAs specific for Nav1.8 SEQ SEQ Duplex sensestrand ID antisense strand ID name sequence (5′-3′) NO: sequence (5′-3′)NO: AL-DP- cauccuaugaaccaauagc 604 gcuauugguucauaggau 605 4461 eTeTgeceT AL-DP- cmaumcmcmumaumgaacm 606 gcumauugguucmaumag 607 4459cmaaumagcmTT-sChol gaugTT AL-DP- umumumumgumcmumaaau 608ugaacucmauuumagacm 609 6980 mgagumumcmaTT-sChol aaaaTT Note: Prefix erepresents 2′-O-(2-methoxyethyl) modified nucleotides

Adult male Sprague-Dawley rats received an injection of CFA into thehindpaw on day 0. Four groups of rats (with 5 rats per group) weretreated starting on day 1 after CFA injection with either siRNA againstNav1.8 (AL-DP-4461, AL-DP-4459, or AL-DP-6980), or PBS. siRNAs againstNav1.8 (AL-DP-4461, AL-DP-4459 or AL-DP-6980) or PBS were administeredby intrathecal bolus injections (10 uL per injection) to the lumbarlevel of the spinal cord twice per day (BID) on days 1, 2 and 3. Tactilehypersensitivity was measured as above, both before CFA injection toassess baseline thresholds, and then on day 4 after CFA and treatmentwith test articles. In rats treated with PBS, tactile hypersensitivitywas pronounced on day 4, as evidenced by reduced paw withdrawalthreshold, as expected. In rats treated with AL-DP-4461 by bolus BID(0.5 mg/bolus), tactile thresholds were moderately normalized on day 4,demonstrating that the Nav1.8 siRNA, AL-DP-4461, is moderatelyefficacious with this dosing paradigm in vivo against CFA-inducedtactile hyperalgesia. Treatment with the Nav1.8 siRNA, AL-DP-4459, bybolus BID (0.15 mg/bolus), resulted in nearly complete normalization oftactile threshold, with all 5 rats demonstrating substantial recovery oftactile thresholds to more than 10.2 g by day 4. In contract, the Nav1.8siRNA AL-DP-6980, by bolus BID (0.15 mg/bolus), did not affect tactilehypersensitivity, showing that not all cholesterol-conjugated siRNAsagainst a target are equally efficacious or potent, even if efficaciouswhen transfected into cultured cells.

These results suggest that cholesterol conjugation enhances in vivoefficacy of siRNAs for neurological disorders, including chronic pain.Furthermore, these results demonstrate that siRNAs targeting Nav1.8,either with or without cholesterol-conjugation, formulated in saline andadministered intrathecally by bolus injection, alleviate CFA-inducedtactile hyperalgesia, and therefore represent a novel approach toproviding effective treatment of clinical inflammatory pain.

Intrathecal Pump or Bolus Administration of siRNAs Against Nav1.8without Transfection Reagent Alleviates Inflammatory Pain

The effect of siRNAs against Nav1.8, formulated in phosphate bufferedsaline (PBS), on complete Freund's adjuvant (CFA)-induced tactilehypersensitivity was evaluated in rats after intrathecal pump infusion(FIG. 7, left) or intrathecal BID bolus injection (FIG. 7, right). Of 3Nav1.8 siRNAs tested by continuous intrathecal pump infusion at 0.4mg/day, 1 siRNA (AL-DP-6050, Table 1) was modestly efficacious againstCFA-induced tactile hypersensitivity. AL-DP-6050, when tested byintrathecal BID bolus injection at 0.5 mg/bolus, was efficacious againstCFA-induced tactile hypersensitivity.

For evaluating effects of siRNAs against NaV1.8 with continuousintrathecal pump infusion, adult male Sprague-Dawley rats received aninjection of CFA into the hindpaw on day 0. Four groups of rats (with 5rats per group) were treated starting on day 1 after CFA injection witheither siRNA against Nav1.8 (AL-DP-6050, AL-DP-6218, or AL-DP-6219), orPBS. In all rats, test articles were intrathecally administered bycontinuous osmotic mini-pump infusion with an infusion rate of 0.5uL/hour, beginning on day 1. siRNAs were infused at 0.4 mg/day. Tactilehypersensitivity was measured as above, both before CFA injection toassess baseline thresholds, and then on day 4 after CFA and treatmentwith test articles (FIG. 7, left). In rats treated with PBS, tactilehypersensitivity was pronounced on day 4, as evidenced by reduced pawwithdrawal threshold, as expected. In rats treated with AL-DP-6050 bycontinuous intrathecal pump infusion (0.4 mg/day), tactile thresholdswere modestly normalized on day 4, demonstrating that the Nav1.8 siRNA,AL-DP-6050, is modestly efficacious with this dosing paradigm in vivoagainst inflammatory pain.

For evaluating effects of siRNAs against NaV1.8 with BID intrathecalbolus injection, adult male Sprague-Dawley rats received an injection ofCFA into the hindpaw on day 0. Two groups of rats (with 4 to 5 rats pergroup) were treated starting on day 1 after CFA injection with eithersiRNA against Nav1.8 (AL-DP-6050, 0.5 mg/bolus) or PBS. Tactilehypersensitivity was assessed as described above. Tactile thresholdswere measured before CFA injection to assess baseline thresholds, andthen on day 4 after CFA injection and treatment with test articles (FIG.7, right). As expected, in rats treated with PBS, tactilehypersensitivity was pronounced on day 4, as evidenced by reduced pawwithdrawal threshold. In rats treated with AL-DP-6050 by BID intrathecalbolus injection (0.5 mg/bolus), tactile thresholds were substantiallynormalized on day 4, demonstrating that the Nav1.8 siRNA, AL-DP-6050, ismodestly efficacious with this dosing paradigm in vivo againstinflammatory pain.

These results further demonstrate that siRNAs targeting Nav1.8 withoutcholesterol-conjugation, formulated in saline and administeredintrathecally by either bolus injection or continuous pump infusion,alleviate CFA-induced tactile hyperalgesia, and therefore represent anovel approach to providing effective treatment of clinical inflammatorypain.

Intrathecal Bolus Administration of siRNAs Against Nav1.8 withoutTransfection Reagent Alleviates Inflammatory Pain

The effect of siRNAs against Nav1.8, formulated in phosphate bufferedsaline (PBS), on complete Freund's adjuvant (CFA)-induced thermalhypersensitivity was evaluated in rats (FIG. 8). Both unconjugated dsRNAAL-DP-6050 (Table 1) and cholesterol-conjugated dsRNA AL-DP-4459 (Table6) were efficacious against CFA-induced thermal hypersensitivity withthe BID bolus intrathecal dosing paradigms evaluated.

Adult male Sprague-Dawley rats received an injection of CFA into thehindpaw on day 0. Three groups of rats (with 4 to 5 rats per group) weretreated starting on day 1 after CFA injection with either siRNA againstNav1.8 (AL-DP-6050 or AL-DP-4459), or PBS. In all rats, test articleswere administered intrathecally by BID bolus injection (10 uL per bolus)beginning on day 1. AL-DP-4459 was dosed at 0.15 mg/bolus whereasAL-DP-6050 was dosed at 0.5 mg/bolus. Thermal hypersensitivity wasmeasured by assessing paw withdrawal latency to a noxious thermalstimulus as described by Hargreaves and colleagues (Hargreaves, K., R.Dubner, et al. (1988) “A new and sensitive method for measuring thermalnociception in cutaneous hyperalgesia” Pain 32:77-88). Latency towithdrawal of a hindpaw in response to noxious radiant heat wasdetermined. A maximal cut-off of 40 sec prevented tissue damage.

Thermal responses were measured before CFA injection to assess baselinethresholds, and then on day 4 after CFA injection and treatment withtest articles. As expected, in rats treated with PBS, thermalhypersensitivity was pronounced on day 4, as evidenced by reduced pawwithdrawal latency. In rats treated with AL-DP-6050 (0.5 mg/bolus) orAL-DP-4459 (0.15 mg/bolus) by intrathecal BID bolus injection, thermallatencies were normalized on day 4, demonstrating that the unconjugatedNav1.8 siRNA, AL-DP-6050, and the cholesterol-conjugated Nav1.8 siRNAAL-DP-4459 are efficacious with this dosing paradigm in vivo againstinflammatory pain.

These results demonstrate that siRNAs targeting Nav1.8, either with orwithout cholesterol-conjugation, formulated in saline and administeredintrathecally by bolus injection, alleviate CFA-induced thermalhyperalgesia, in addition to tactile hyperalgesia (above), and thereforerepresent a novel approach to providing effective treatment of multipletypes of hyperalgesia in clinical inflammatory pain.

Intrathecal Bolus Administration of Cholesterol-Conjugated siRNA AgainstNav1.8 without Transfection Reagent Alleviates Neuropathic Pain

The effect of AL-DP-4459 (Table 6) against Nav1.8, formulated inphosphate buffered saline (PBS), on spinal nerve ligation (SNL)-inducedtactile and thermal hypersensitivity was evaluated in rats (FIG. 9).With the bolus intrathecal dosing paradigm evaluated (0.15 mg/bolus,BID), AL-DP-4459 was efficacious against SNL-induced tactile and thermalhypersensitivity.

Adult male Sprague-Dawley rats received unilateral ligation of the L5and L6 spinal nerves on day 0 (SNL surgery). Three groups of rats (with6 to 8 rats per group) were treated starting on day 3 after SNL surgeryby intrathecal administration of either the siRNA AL-DP-4459 againstNav1.8, or PBS. In 2 of these groups, AL-DP-4459 or PBS was administeredby intrathecal bolus injections (5 uL per injection) to the lumbar levelof the spinal cord twice per day (BID) on post-SNL days 3 through 7. Inone group of rats, AL-DP-4459 was intrathecally administered bycontinuous osmotic mini-pump infusion at 0.18 mg/day, with an infusionrate of 0.5 uL/hour, on post-SNL days 3 through 7. Tactile and thermalhypersensitivities were assessed as described above.

Tactile (FIG. 9, left) and thermal (FIG. 9, right) responses weremeasured before SNL surgery to assess baseline responses (BL), and onpost-SNL day 3 before treatment with test articles to verify thattactile and thermal hyperalgesia had developed fully. In rats treatedwith PBS, tactile and thermal hypersensitivities were pronounced, asexpected, on post-SNL days 3, 5 and 7 as evidenced by reduced pawwithdrawal thresholds and reduced thermal latencies, respectively. TheNav1.8 siRNA AL-DP-4459, when intrathecally administered by continuouspump infusion at 0.18 mg/day, did not affect tactile or hypersensitivitysignificantly over the time-frame shown. In contrast, in rats treatedwith AL-DP-4459 by bolus BID (0.15 mg/bolus), tactile thresholds andthermal latencies were substantially normalized on post-SNL day 7 (day 5of treatment), demonstrating that the Nav1.8 siRNA, AL-DP-4459, isefficacious in vivo against SNL-induced tactile and thermalhyperalgesia.

These results demonstrate that intrathecally administeredcholesterol-conjugated siRNAs targeting Nav1.8, formulated in saline,are efficacious against experimental nerve injury-induced chronic painin rats, and therefore, represent a novel approach to providingeffective treatment of clinical neuropathic pain.

Unconjugated and Cholesterol-Conjugated siRNAs Targeting NaV1.8 areStable in Human Cerebrospinal Fluid at 37° C.

To determine the stability in human cerebrospinal fluid (CSF) ofunconjugated and cholesterol-conjugated siRNAs targeting NaV1.8, siRNAduplexes were incubated in human CSF for 48 hours at 37° C., and thesingle strands were measured by quantitative ion exchangechromatography. In this example, unconjugated siRNAs AL-DP-6050,AL-DP-6209, AL-DP-6217, AL-DP-6218 and AL-DP-6219 (Table 1), andcholesterol-conjugated siRNA AL-DP-4459 (Table 6) were evaluated. 30 μlof human CSF was mixed with 3 μl of 50 μM siRNA (150 pmole/well) in a96-well plate, sealed to avoid evaporation, and incubated for 1 to 48hours at 37° C. Incubation in 30 μl PBS for 48 hours at 37° C. served asa control for nonspecific degradation. Reactions were stopped by theaddition of 4 μl proteinase K (20 mg/ml) and 25 μl of proteinase Kbuffer, and incubation of this mixture for 20 min at 42° C. Samples werespin filtered through a 0.2 μm 96-well filter plate at 3000 rpm for 20min. Incubation wells were washed with 50 μl Millipore water twice andthe combined washing solutions were spin filtered also. A 5 μl aliquotof 50 μM 40-mer RNA was added to each sample to act as an internalstandard (IS) for normalization of volume changes in the filtrationvolume. Samples were analyzed by ion exchange HPLC under denaturingconditions.

1. HPLC System for analysis of cholesterol-conjugated siRNA AL-DP-4459

-   -   Column: Dionex DNAPac PA200 (4×250 mm analytical column)    -   Temp.: 80° C. (denaturing conditions)    -   Flow: 1 ml/min    -   Injection: 50 ul    -   Detection: 260 (reference wavelength 600 nm)    -   HPLC Eluent A: 25 mM TRIS-HCl; 1 mM EDTA; 50% ACN; pH=8    -   HPLC Eluent B: 600 mM NaBr in A    -   Gradient table:

Time % A % B 0.00 min 80 20 1.00 min 80 20 15.0 min 30 70 15.5 min 0 10017.5 min 0 100 18.0 min 80 20 21.0 min 80 20

2. HPLC System for unconjugated siRNAs AL-DP-6050, 6209, 6217, 6218,6219

-   -   Column: Dionex DNAPac PA200 (4×250 mm analytical column)    -   Temp.: 30° C. (denaturing conditions by pH=11)    -   Flow: 1 ml/min    -   Injection: 50 ul    -   Detection: 260 nm (reference wavelength 600 nm)    -   HPLC Eluent A: 20 mM Na₃PO₄ in 10% ACN; pH=11    -   HPLC Eluent B: 1 M NaBr in A    -   Gradient table:

Time % A % B 0.00 min 75 25 1.00 min 75 25 21.0 min 40 60 21.5 min 0 10023.0 min 0 100 24.5 min 75 25 28.0 min 75 25

Under the denaturing IEX-HPLC conditions, the duplexes eluted as twoseparated single strands. The internal standard eluted at higherretention times than the single strands of the duplex, and did notinterfere with the analysis.

For every injection, the chromatograms were integrated automatically bythe Dionex Chromeleon 6.60 HPLC software, but were adjusted manually asnecessary. All peak areas were corrected by the following equation tothe internal standard (IS) peak:

CF _((1S))=100/PeakArea_((IS)); Corrected PeakArea_((s/as))=CF*PeakArea_((a/as))

The %-values for the remaining intact FLP at all time points arecalculated by the following equation:

%-FLP _((s/as))=(Corrected PeakArea_((s/as))/CorrectedPeakArea_((s/as); t=0 min))*100%

All values were normalized to the incubation at t=0 min.

The results for the unconjugated siRNAs are shown in FIG. 10. After 48hours incubation at 37° C. in PBS (PBS-48), all five siRNAs werecompletely stable, with no loss detectable. After 48 hours incubation at37° C. in human CSF, three (AL-DP-6050, AL-DP-6209 and AL-DP-6217) ofthe five siRNAs exhibited less than 20% loss, whereas two (AL-DP-6218and AL-DP-6219) of the siRNAs exhibited greater than 50% loss. Theresults for the cholesterol-conjugated siRNA AL-DP-4459 are shown inFIG. 11. After 48 hours incubation at 37° C. in PBS (PBS-48), AL-DP-4459was completely stable, with no loss detectable. When assessed forstability in human CSF, AL-DP-4459 was found to be stable at 37° C. for48 hours, with less than 20% variation detected over this time period.

Unconjugated and Cholesterol-Conjugated siRNAs Targeting Nav1.8 ReduceNav1.8 mRNA in a Dose-Dependent Manner in Primary Rat Sensory NeuronalCultures

Another effective siRNA (AL-DP-6050, Table 1) against Nav1.8 from thesingle dose screen in primary cultures of rat dorsal root ganglion cellsand its cholesterol conjugate AL-DP-4459 (Table 6) were furthercharacterized for dose dependence. For the dose response curves,transfections were performed using Lipofectamine 2000 (see below) withthe following concentrations of siRNA: 200, 80, 32, 12.8, 5.12, 2.05,0.82 and 0.33 nM (FIG. 12).

DRG neurons were transfected 24 h post-plating. For each well, 0.4 μlLipofectamine™ 2000 (Invitrogen Corporation, Carlsbad, Calif.) was usedand transfections were performed according to the manufacturer'sprotocol. Specifically, the siRNA: Lipofectamine™ 2000 complexes wereprepared as follows. The appropriate amount of siRNA was diluted inOpti-MEM I Reduced Serum Medium and mixed gently. The Lipofectamine™2000 was vortexed before use; then for each well of a 96 well plate, 0.4ul Lipofectamine™ 2000 was diluted in 25 μl of Opti-MEM I Reduced SerumMedium, mixed gently and incubated for 5 minutes at room temperature.After the 5 minute incubation, 1 ul of the diluted siRNA was combinedwith the diluted Lipofectamine™ 2000 (total volume, 26.4 μl). Thecomplex was mixed gently and incubated for 20 minutes at roomtemperature to allow the siRNA: Lipofectamine™ 2000 complexes to form.Then 100 μl of serum-free F12-HAM's Medium containing glutamine(Invitrogen Gibco, Carlsbad Calif., USA) supplemented with 50 ng/ml NGF2.5S (Promega Corp., Madison Wis., USA) and 1:50 B27 supplement(Invitrogen Gibco, Carlsbad Calif., USA) was added to the transfectioncomplexes, and mixing was achieved by gently pipetting up and down. Thegrowth medium was removed from the DRG cells and 100 μl of the abovetransfection complex mixture was added to each well of a 96-well plate.After 20 h of incubation at 37° C., 5% CO₂ in a humidified incubator,supernatant was removed from the cells, fresh F12-HAM's mediumcontaining glutamine supplemented with 5% FBS, 5% horse serum (bothInvitrogen Gibco, Carlsbad Calif., USA), 50 ng/ml mouse NGF 2.5S(Promega Corp., Madison Wis., USA) and 1:100 Penicillin/Streptomycin(Invitrogen Gibco, Carlsbad Calif., USA) was added. The cells wereincubated for another 20-24 h at 37° C., 5% CO₂ in a humidifiedincubator, and Nav1.8 mRNA was quantified by the bDNA assay, asdescribed previously.

FIG. 12 provides the result for the selected siRNA AL-DP-6050 and itscholesterol conjugate AL-DP-4459 from a dose response experiment.AL-DP-6050 and its cholesterol conjugate AL-DP-4459 inhibited Nav1.8mRNA expression relative to alpha-synuclein (SNCA) in a dose-dependentmanner, with maximal inhibitions of −40% at >30 nM siRNA, in thisexperiment.

Intrathecal Bolus Administration of Unconjugated siRNA Against Nav1.8without Transfection Reagent Alleviates Neuropathic Pain

The effect of AL-DP-6050 (Table 1) against Nav1.8, formulated inphosphate buffered saline (PBS), on spinal nerve ligation (SNL)-inducedthermal hypersensitivity was evaluated in rats (FIG. 13). With the bolusintrathecal dosing paradigm evaluated (0.15 mg/bolus, BID), AL-DP-6050was efficacious against SNL-induced thermal hypersensitivity.

Adult male Sprague-Dawley rats received unilateral ligation of the L5and L6 spinal nerves on day 0 (SNL surgery). Two groups of rats (with 8rats per group) were treated starting on day 3 after SNL surgery byintrathecal bolus injection with either the siRNA AL-DP-6050 againstNav1.8, or PBS. Intrathecal bolus injections (0.15 mg in 5 uL perinjection) were administered to the lumbar level of the spinal cordtwice per day (BID) on post-SNL days 3 through 7. Thermalhypersensitivity was assessed as described above.

Thermal responses were measured before SNL surgery to assess baselineresponses (BL), and on post-SNL day 3 before treatment with testarticles to verify that thermal hyperalgesia had developed fully. Inrats treated with PBS, thermal hypersensitivity was pronounced, asexpected, on post-SNL days 3, 5 and 7 as evidenced by reduced thermallatencies. In contrast, in rats treated with AL-DP-6050 by bolus BID(0.15 mg/bolus), thermal latencies were substantially normalized onpost-SNL days 5 (day 3 of treatment) and 7 (day 5 of treatment),demonstrating that the Nav1.8 siRNA, AL-DP-6050, is efficacious in vivoagainst SNL-induced thermal hyperalgesia.

These results demonstrate that siRNAs targeting Nav1.8, formulated insaline and administered by intrathecal bolus injection, are efficaciousagainst experimental nerve injury-induced chronic pain in rats, andtherefore, represent a novel approach to providing effective treatmentof clinical neuropathic pain.

Intrathecal Bolus Administration of ND98-2.7 Liposomal Formulation ofsiRNA Against Nav1.8 Alleviates Neuropathic Pain

The effect of AL-DP-6050 (Table 1) against Nav1.8, formulated inND98-2.7 (described below), and administered by intrathecal bolusinjection, on spinal nerve ligation (SNL)-induced thermalhypersensitivity was evaluated in rats (FIG. 14). With the bolusintrathecal dosing paradigm evaluated (5 micrograms/bolus, daily),AL-DP-6050 was efficacious against SNL-induced thermal hypersensitivity.

Adult male Sprague-Dawley rats received unilateral ligation of the L5and L6 spinal nerves on day 0 (SNL surgery). Two groups of rats (with 6rats per group) were treated starting on day 3 after SNL surgery byintrathecal bolus injection with either the siRNA AL-DP-6050 againstNav1.8, formulated in ND98-2.7, or PBS. Intrathecal bolus injections (5micrograms in 5 uL per injection) were administered to the lumbar levelof the spinal cord daily on post-SNL days 3 through 5. Thermalhypersensitivity was assessed as described above.

Thermal responses were measured before SNL surgery to assess baselineresponses (BL), and on post-SNL day 3 before treatment with testarticles to verify that thermal hyperalgesia had developed fully. Inrats treated with PBS, thermal hypersensitivity was pronounced, asexpected, on post-SNL days 3, 5, 7 and 10 as evidenced by reducedthermal latencies. In contrast, in rats treated with AL-DP-6050formulated in ND98-2.7 liposomes, by intrathecal bolus daily injection(5 micrograms/bolus), thermal latencies were substantially normalized onpost-SNL day 5 (day 3 of treatment), demonstrating that the Nav1.8siRNA, AL-DP-6050, formulated in ND98-2.7 liposomes and administered byintrathecal injection, is efficacious in vivo against SNL-inducedthermal hyperalgesia. Moreover, the dose level of siRNA required forefficacy with the ND98-2.7 liposomal formulation (5 micrograms per day)was much lower than that observed for efficacy with the PBS formulation(300 micrograms per day).

These results demonstrate that siRNAs targeting Nav1.8, administered byintrathecal bolus injection and formulated in ND98-2.7 liposomes, areefficacious against experimental nerve injury-induced chronic pain inrats, and therefore, represent a novel approach to providing effectivetreatment of clinical neuropathic pain.

Intravenous Bolus and Continuous Pump Administration of ND98-2.7Liposomal Formulation of siRNA Against Nav1.8 Alleviates NeuropathicPain

The effect of AL-DP-6050 (Table 1) against Nav1.8, formulated inND98-2.7, and administered by intravenous bolus injection or intravenouscontinuous pump infusion, on spinal nerve ligation (SNL)-induced thermalhypersensitivity was evaluated in rats (FIG. 14). With the bolusintravenous dosing paradigm evaluated (daily 0.5 mg/bolus, equivalent toapproximately 2 mg/kg/bolus), and with the continuous intravenous pumpparadigm evaluated (0.24 mg/day, equivalent to approximately 1mg/kg/day), AL-DP-6050, formulated in ND98-2.7 liposomes, wasefficacious against SNL-induced thermal hypersensitivity.

Adult male Sprague-Dawley rats received unilateral ligation of the L5and L6 spinal nerves on day 0 (SNL surgery). Two groups of rats (with 6rats per group) were treated starting on day 3 after SNL surgery byintravenous bolus injection or intravenous continuous pump infusion withthe siRNA AL-DP-6050 against Nav1.8, formulated in ND98-2.7. Intravenousbolus injections (0.5 mg in 1 mL per bolus) were administered daily onpost-SNL days 3 through 5. Intravenous continuous pump infusion (10uL/hour, equivalent to 0.24 mg/day) was administered on post-SNL days 3through 10 via a cannula in the jugular vein. Thermal hypersensitivitywas assessed as described above.

Thermal responses were measured before SNL surgery to assess baselineresponses (BL), and on post-SNL day 3 before treatment with testarticles to verify that thermal hyperalgesia had developed fully.Thermal hypersensitivity was pronounced, as expected, on post-SNL day 3,before treatment, as evidenced by reduced paw withdrawal latencies. Incontrast, in rats treated with AL-DP-6050 formulated in ND98-2.7liposomes, by intravenous bolus daily injection (0.5 mg/bolus), thermallatencies were substantially normalized on post-SNL day 5 (day 3 oftreatment), and post-SNL day 7 (2 days after the last intravenous bolustreatment) demonstrating that the Nav1.8 siRNA, AL-DP-6050, formulatedin ND98-2.7 liposomes and administered by intravenous bolus injection,is efficacious in vivo against SNL-induced thermal hyperalgesia.Furthermore, in rats treated with AL-DP-6050 formulated in ND98-2.7liposomes, by intravenous continuous pump infusion (0.24 mg/day),thermal latencies were substantially normalized on post-SNL days 5 (day3 of treatment), 7 (day 5 of treatment) and 10 (day 8 of treatment).

These results demonstrate that siRNAs targeting Nav1.8, formulated inND98-2.7 liposomes, and administered by intravenous bolus injection orintravenous continuous pump infusion, are efficacious againstexperimental nerve injury-induced chronic pain in rats, and therefore,represent a novel approach to providing effective treatment of clinicalneuropathic pain. Moreover, these results demonstrate that ND98-2.7liposomal formulation of siRNA can provide effective delivery of siRNAto sensory neurons of the dorsal root ganglion, with systemicadministration.

Formulation Procedure

The lipidoid ND98•4HCl (MW 1487), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNAnanoparticles.

Stock solutions of each in ethanol were prepared: ND98 (FIG. 15, ND98Isomer I), 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100mg/mL. ND98, Cholesterol, and PEG-Ceramide C16 stock solutions were thencombined in a 42:48:10 molar ratio. Combined lipid solution was mixedrapidly with aqueous siRNA (in sodium acetate pH 5) such that the finalethanol concentration was 35-45% and the final sodium acetateconcentration was 100-300 mM. Lipid-siRNA nanoparticles formedspontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture was in some casesextruded through a polycarbonate membrane (100 nm cut-off) using athermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In othercases, the extrusion step was omitted. Ethanol removal and simultaneousbuffer exchange was accomplished by either dialysis or tangential flowfiltration. Buffer was exchanged to phosphate buffered saline (PBS) pH7.2.

Characterization of Formulations

Formulations prepared by either the standard or extrusion-free methodare characterized in a similar manner. Formulations are firstcharacterized by visual inspection. They should be whitish translucentsolutions free from aggregates or sediment. Particle size and particlesize distribution of lipid-nanoparticles are measured by dynamic lightscattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particlesshould be 20-300 nm, and ideally, 40-100 nm in size. The particle sizedistribution should be unimodal. The total siRNA concentration in theformulation, as well as the entrapped fraction, is estimated using a dyeexclusion assay. A sample of the formulated siRNA is incubated with theRNA-binding dye Ribogreen (Molecular Probes) in the presence or absenceof a formulation disrupting surfactant, 0.5% Triton-X100. The totalsiRNA in the formulation is determined by the signal from the samplecontaining the surfactant, relative to a standard curve. The entrappedfraction is determined by subtracting the “free” siRNA content (asmeasured by the signal in the absence of surfactant) from the totalsiRNA content. Percent entrapped siRNA is typically >85%.

dsRNA Expression Vectors

In another aspect of the invention, Nav1.8 specific dsRNA molecules thatmodulate Nav 1.8 gene expression activity are expressed fromtranscription units inserted into DNA or RNA vectors (see, e.g.,Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al.,International PCT Publication No. WO 00/22113, Conrad, International PCTPublication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Thesetransgenes can be introduced as a linear construct, a circular plasmid,or a viral vector, which can be incorporated and inherited as atransgene integrated into the host genome. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. In apreferred embodiment, a dsRNA is expressed as an inverted repeat joinedby a linker polynucleotide sequence such that the dsRNA has a stem andloop structure.

The recombinant dsRNA expression vectors are preferably DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector of the invention may be a eukaryotic RNA polymerase I (e.g.ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter oractin promoter or U1 snRNA promoter) or preferably RNA polymerase IIIpromoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter,for example the T7 promoter, provided the expression plasmid alsoencodes T7 RNA polymerase required for transcription from a T7 promoter.The promoter can also direct transgene expression to the pancreas (see,e.g. the insulin regulatory sequence for pancreas (Bucchini et al.,1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Preferably, recombinant vectors capable of expressing dsRNA moleculesare delivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single Nav1.8 gene or multiple Nav1.8 genes over a period of a weekor more are also contemplated by the invention. Successful introductionof the vectors of the invention into host cells can be monitored usingvarious known methods. For example, transient transfection can besignaled with a reporter, such as a fluorescent marker, such as GreenFluorescent Protein (GFP). Stable transfection. of ex vivo cells can beensured using markers that provide the transfected cell with resistanceto specific environmental factors (e.g., antibiotics and drugs), such ashygromycin B resistance.

The Nav1.8 specific dsRNA molecules can also be inserted into vectorsand used as gene therapy vectors for human patients. Gene therapyvectors can be delivered to a subject by, for example, intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) or bystereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad.Sci. USA 91:3054-3057). The pharmaceutical preparation of the genetherapy vector can include the gene therapy vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.,retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting theexpression of a human Nav1.8 gene in a cell, wherein said dsRNAcomprises at least two sequences that are complementary to each otherand wherein a sense strand comprises a first sequence and an antisensestrand comprises a second sequence comprising a region ofcomplementarity which is substantially complementary to at least a partof a mRNA encoding Nav1.8, and wherein said region of complementarity isless than 30 nucleotides in length and wherein said dsRNA, upon contactwith a cell expressing said Nav1.8, inhibits expression of said Nav1.8gene by at least 20%.
 2. The dsRNA of claim 1, wherein said firstsequence is selected from the group consisting of Tables 1, 4 and 6 andsaid second sequence is selected from the group consisting of Tables 1,4 and
 6. 3. The dsRNA of claim 1, wherein said dsRNA comprises at leastone modified nucleotide.
 4. The dsRNA of claim 2, wherein said dsRNAcomprises at least one modified nucleotide.
 5. The dsRNA of claim 3,wherein said modified nucleotide is chosen from the group of: a2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.
 6. ThedsRNA of claim 3, wherein said modified nucleotide is chosen from thegroup of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-O-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.
 7. A cellcomprising the dsRNA of claim
 1. 8. A pharmaceutical composition forinhibiting the expression of the Nav1.8 gene in an organism, comprisinga dsRNA and a pharmaceutically acceptable carrier, wherein the dsRNAcomprises at least two sequences that are complementary to each otherand wherein a sense strand comprises a first sequence and an antisensestrand comprises a second sequence comprising a region ofcomplementarity which is substantially complementary to at least a partof a mRNA encoding Nav1.8, and wherein said region of complementarity isless than 30 nucleotides in length and wherein said dsRNA, upon contactwith a cell expressing said Nav1.8, inhibits expression of said Nav1.8gene by at least 20%.
 9. The pharmaceutical composition of claim 8,wherein said first sequence of said dsRNA is selected from the groupconsisting of Tables 1, 4 and 6 and said second sequence of said dsRNAis selected from the group consisting of Tables 1, 4 and
 6. 10. Thepharmaceutical composition of claim 9, wherein said composition isformulated for administration selected from the group consisting ofintrathecal infusion or injection, or intravenous infusion or injection.11. A method for inhibiting the expression of the Nav1.8 gene in a cell,the method comprising: (a) introducing into the cell a double-strandedribonucleic acid (dsRNA), wherein the dsRNA comprises at least twosequences that are complementary to each other and wherein a sensestrand comprises a first sequence and an antisense strand comprises asecond sequence comprising a region of complementarity which issubstantially complementary to at least a part of a mRNA encodingNav1.8, and wherein said region of complementarity is less than 30nucleotides in length and wherein said dsRNA, upon contact with a cellexpressing said Nav1.8, inhibits expression of said Nav1.8 gene by atleast 20%; and (b) maintaining the cell produced in step (a) for a timesufficient to obtain degradation of the mRNA transcript of the Nav1.8gene, thereby inhibiting expression of the Nav1.8 gene in the cell. 12.The method of claim 11, wherein said first sequence of said dsRNA isselected from the group consisting of Tables 1, 4 and 6 and said secondsequence of said dsRNA is selected from the group consisting of Tables1, 4 and
 6. 13. A method of treating, preventing or managing paincomprising administering to a patient in need of such treatment,prevention or management a therapeutically or prophylactically effectiveamount of a dsRNA, wherein the dsRNA comprises at least two sequencesthat are complementary to each other and wherein a sense strandcomprises a first sequence and an antisense strand comprises a secondsequence comprising a region of complementarity which is substantiallycomplementary to at least a part of a mRNA encoding Nav1.8, and whereinsaid region of complementarity is less than 30 nucleotides in length andwherein said dsRNA, upon contact with a cell expressing said Nav1.8,inhibits expression of said Nav1.8 gene by at least 20%.
 14. The methodof claim 13, wherein said first sequence of said dsRNA is selected fromthe group consisting of Tables 1, 4 and 6 and said second sequence ofsaid dsRNA is selected from the group consisting of Tables 1, 4 and 6.15. The method of claim 14, wherein said pain is selected from the groupconsisting of neuropathic pain and inflammatory pain.
 16. A vector forinhibiting the expression of the Nav1.8 gene in a cell, said vectorcomprising a regulatory sequence operably linked to a nucleotidesequence that encodes at least one strand of a dsRNA, wherein one of thestrands of said dsRNA is substantially complementary to at least a partof a mRNA encoding Nav1.8 and wherein said dsRNA is less than 30 basepairs in length and wherein said dsRNA, upon contact with a cellexpressing said Nav1.8, inhibits the expression of said Nav1.8 gene byat least 20%.
 17. The vector of claim 16, wherein said first sequence ofsaid dsRNA is selected from the group consisting of Tables 1, 4 and 6and said second sequence of said dsRNA is selected from the groupconsisting of Tables 1, 4 and
 6. 18. A cell comprising the vector ofclaim
 16. 19. A cell comprising the vector of claim 17.